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N. Tabacum x N. Rustica

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deluxestogie

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The report from a panel of tobacco tasting experts reminds me of the report from such a panel (in the early 20th century) of tobacco that was routinely treated with Paris Green, to kill insect pest. The company's conclusion was that the cigarettes made from tobacco treated with Paris Green tastes really good, and burns beautifully. So the researcher strongly recommended the dusting of Paris Green onto growing tobacco to minimize pests and enhance the smoking quality.

The punchline is that Paris Green is a copper-arsenic compound that is highly toxic to all mammals. But...it tastes so good!

Bob
 

OldDinosaurWesH

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Bob:

Your response talking about Paris Green engenders a plant pathology story on my part. We learned about this in Plant Pathology class in college.

During the nineteenth century, there was a grape grower in France who's vineyard was next to a main road. During the harvest season, as the school children walked by the vineyard, they would stop and pick and eat his grapes. In order to stop the school children from eating his grapes he mixed up a spray made up of Copper Sulfate and water and sprayed it all over the grape vines to make them unappealing. Meanwhile, a blight disease (fungus) went throughout France and wiped out all the grape vines...Except for those vines that had been sprayed with copper sulfate. This was the accidental discovery of the world's first fungicide. There is still a class of fungicides in use today known as the "Organic Coppers." Copper, Silver, and Gold, arrayed vertically above one another on the periodic table, are all highly anti-microbial. Hospitals are switching to copper door handles because of copper's anti-microbial characteristics.

The "Organic Arsenicals" were a class of mostly insecticides that were used heavily in the early 20th century. Someone finally figured out that these weren't good for people and that the Arsenic accumulated in the soils. Arsenic is one of the what we used to call "semi-metals". Now I believe they call it a "transition element". Pure metallic Arsenic has all the properties of a metal except one...It won't conduct electricity. Arsenic is chemically related to sulfur, and your body will take it in and bind it to the same molecular chains where sulfur would fit. I believe there is a highly technical term for this phenomenon. "Bad," comes to mind. Also, Arsenic is a relatively common element. There is quite a bit of it out there in nature.

That's my college lecture for the day. I have more if anyone is interested.

Wes H.
 

Tutu

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I've got my hands on a number of other articles relating to this subject matter. Most of them are usually paid for when acquired. The first article I'd like to discuss is written by two Japanese and a Korean. It's really interesting stuff, but its quite technical as well. Let me walk you through it.

The diversity of interspecific pollen-pistil incongruity in Nicotiana

As I mentioned, this one is quite in-depth when it comes to technical aspects. But it's exactly what I was looking for. Basically, the researchers investigate what happens to the pollen tubes when attempting to make three different hybrids. For all three hybrids, N. tabacum functions as the female parent. Three other species within Nicotiana provide the pollen. They are N. repanda, N. rustica and N. trigonophylla. For this current thread we are mainly interested in the cross with N. rustica. That being said, it's also quite interesting to see how the behaviour of pollen is different in different species. So we'll have a look at all of them.

The first given from the article is that the pollen tubes, which act as the conduit to transport the male gamete from the pollen to the female ovary, are unilaterally inhibited by the pistils of N. tabacum. So where the pollen of N. tabacum pollen on a N. tabacum pistil would send the gamete straight into the ovary, this is hindered when the pollen is not from N. tabacum but from one of the other mentioned species. This is what makes it difficult to create hybrids.

The authors refer to another article which I will come to in a later post, that holds that there are many combinations of hybrids in which pollen tubes only reach the ovary in one direction of the cross, but not its reciprocal. We had heard this before. This is what many report to be the case in N. rustica x N. tabacum versus the other way around, N. tabacum x N. rustica. In this current article we will find out why this is the case.

So this research revolves around measurements of pollen tubes. Pollen tube morphology and elongation patterns. To have material for comparison they start with looking at self-fertilization. After that they go for the hybrids. It's too bad that these aren't colour shots, but they're good enough to make things out.


Pollen tubes.jpg

It might take a while to adjust to these microscope photographs. They are all pollen tubes, and they should be straight lines, headed for the ovary. When self fertilized, N. tabacum, N. repanda, N. rustica and N. trigonophylla all portray straight lines on the pictures. The hybrid pollen tubes do not. For our focus here, picture G is the most interesting as it shows N. tabacum x N. rustica. The direction of the tubes is winding sort of a winding trend. This is not good, because it limits the length in which the tube can stretch itself. It needs a minimum length in order to reach the ovary. There is another figure in this paper which shows a longitudinal cross section of a pollen tube of the N. tabacum x N. rustica hybrid and it shows a pollen tube elongating backward. You don't reach ovaries that way, that's for sure.

So let's have a little look at the graphical trajectory of pollen tubes.

Pollen graph.jpg

As described, the first graph shows the average length of pollen tubes in self fertilizing species. As you can see, N. tabacum pollen tubes travel furthest. N. repanda finishes a good second. It takes a further distance to reach the N. tabacum ovary. So when we're looking at the behaviour of pollen tubes in the cross fertilizations we see that on average, the pollen tubes of other species do not reach the required length. We also see that they do behave different compared to fertilizing one of their own species. For the first 12 hours N. rustica pollen tubes elongate even faster than N. tabacum fertilizing itself. However, after reaching 23 mm on average, there wasn't much growth detected. Some pollen would swell, circle about, or even move backward.


Inhibitor type.jpg

The authors give each style of inhibiting a label. For N. rustica to fertilize N. tabacum, they call it the Winding type because of the form of movement by the pollen tubes. N. repanda is the stigma type, as these pollen remained on the stigma. N. trigomophylla is the delay type. But there is yet another thing.

Development stages.jpg

They look at the pollen tubes in further detail, namely, pollination at different ages of the female flower pistil. They divide these stages into four. For all three inhibiting types it became apparent that the inhibition is strengthened during maturation of the pistil. In other words, the older the flower, the less changes of pollen tubes reaching the ovary. In N. tabacum x N. rustica, pollen tubes were found in every ovary when crossed at stage 1 of the female flower stage. Then, with maturation of the flower, crossing results in fewer ovaries containing pollen. So immature pistils of N. tabacum are less inhibitory to pollen from N. rustica. The same is true for the other varieties replacing the male parent. This is interesting stuff, as for crossing in the field we need to work with very young pistils, as to make sure that the flower has to prevent self pollination. It seems that it raises the chance of success, if anything.

Hybrid tube.jpg

Then there's a last little experiment which is where the authors look at pollen fertilizing F[SUB]1[/SUB] hybrids. However, as an F[SUB]1[/SUB] hybrids they are using N. rustica x N. tabacum, not the N. tabacum x N. rustica. That last hybrid would have been more interesting to experiment on, as we could maybe have predicted whether it is easier to pollinate the hybrid than it is to make the hybrid in the first place.
 

Charly

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Great stuff !
I hope it will help you creating your own crosses !
Good luck ;)
 

Tutu

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I hope so too. My question for the researchers would be how much the deviation from the averages is in the data displayed in the graphs. If N. rustica pollen tubes travel 23mm on average, what are the outliers? Would the maximum outlier exceed the required 36 mm or not. I suppose it's a long shot. Anyway, it does bring us a little bit closer to a better understanding of the barriers to make hybrids in a natural way.
 

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In the graphs, the vertical bar at each mean data point indicates either the variance or 1 or 2 standard deviations. I believe table 2 may be raw data. Table 3 directly indicates some margin of error.

I have a further consideration to add to this thread discussion, in light of the pollen tube length research. Both the absolute and relative length of the pistil and anthers differs in specific N. tabacum varieties. Back in 2012, I noted a marked difference in the length of the pistil the Oriental variety Mutki, vs the Oriental variety Balikesir. (My original interest was in how this might impact the percentage of self-pollination vs open-pollination.) But...A short pistil means a better chance for a slow and confused pollen tube to reach its goal.

Garden20120804_438_Balikesir_blossom_400.jpg


Garden20120830_466_Balikesir_shortPistil_300.jpg
Garden20120830_467_Mutki_tallPistil_300.jpg


Bob
 

Tutu

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You are completely right about the vertical bars. Not sure how I could have missed that. I suppose they indicate one standard deviation indeed.

However, I am very, very interested in the other consideration you are raising here. While the deviation in pollen tube might affect the chances of fertilization, a deviation in pistil size might be an even bigger contributor. I had not thought of this at all. I guess these subtle differences are in correlation with growing experience, and with having an eye for detail. This excites me very much. The difference in pistil size really looks substantial in the picture.

I need to ask though. Do you reckon that the Mutki had a relatively large pistil in comparison with most other varieties? Or is the Balikesir really quite short? What I mean to ask is, your average N. tabacum, would you say it resembles which pistil? In any which way, it would be interesting to learn if this is a specific trait that the Balikesir variety has.

You really might be on to something here. This may be the long lasting dream for confused pollen tubes come true.
 

deluxestogie

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The following is a selection of the clearest blossom views that I have located among my photos. The first (unlabeled) is of Columbia Garcia from GRIN. [I'm sure that I have a bunch more, but they will take a substantial effort to find.]

ColumbianGarcia_blossom_GRINphoto.JPG


Garden20120709_318_Papante_Blossom_300.jpg


Garden20120709_321_MtPima_Blossom_300.jpg


Garden20130810_873_Prilep_P66-9_7_blossom_400.jpg


Garden20140715_1353_cyprusOriental_blossom_400.jpg


Garden20140719_1359_Chichicaste712_blossom_400.jpg


Garden20140724_1366_LongRed_blossom_400.jpg


Garden20140827_1489_CyprusLatakia_blossom_400.jpg


Garden20170726_2906_PrancakN1_blossom_600.jpg


An interesting theme is that the Oriental varieties tend to sport shorter pistils than the others. I'll have to locate additional photos, or do more observations next season, to determine if that is true.

Bob
 

deluxestogie

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Hasse, thanks for posting that. My German is inadequate for the task. It is difficult reading, even using Google Translate (one section at a time), since it is unable to translate many of the words, and mis-translates some. For example Narbe is translated as "scar", instead of "stigma".

A curious observation is that the stamen are always of different lengths within a single blossom, with at least one of them supposedly always being shorter than the pistil, and at least one of them longer than the pistil. And that this betrays the pentagonal symmetry of the blossom. I was unable to find a discussion of the pistil lengths.

The descriptions of the genetic sources of Mont Calme Jaune and Mont Calm Brun are more detailed than any I've found before (and with Amersfoort in the mix!).

Bob
 

deluxestogie

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Those are big radiation doses. If I recall correctly, the acute lethal dose of ionizing radiation for humans (LD[sub]50[/sub]) is in the ballpark of 600 rads. They're using 10 times that.

"...irradiation of pollen may also lead to genetic changes resulting in the inactivation of zygotic lethals." Not to mention entirely unpredictable genetic mutations.

While it is possible to store (and presumably ship) Nicotiana pollen for several weeks, and still maintain viability, it's vigor is significantly reduced, affecting its ability to successfully produce a pollen tube.

NicotianaPollenGermination_GRAPH.JPG

https://link.springer.com/article/10.1007/BF00226109

So, unless you do the irradiation as well as the fertilization (the actual cross) in the same location, the "benefits" of ionizing radiation on pollen tube growth may be lost if the resulting pollen is shipped or stored prior to use.

And do you really want to mess with GMO tobacco?

Bob
 

ChinaVoodoo

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Those are big radiation doses. If I recall correctly, the acute lethal dose of ionizing radiation for humans (LD[sub]50[/sub]) is in the ballpark of 600 rads. They're using 10 times that.

"...irradiation of pollen may also lead to genetic changes resulting in the inactivation of zygotic lethals." Not to mention entirely unpredictable genetic mutations.

While it is possible to store (and presumably ship) Nicotiana pollen for several weeks, and still maintain viability, it's vigor is significantly reduced, affecting its ability to successfully produce a pollen tube.

NicotianaPollenGermination_GRAPH.JPG

https://link.springer.com/article/10.1007/BF00226109

So, unless you do the irradiation as well as the fertilization (the actual cross) in the same location, the "benefits" of ionizing radiation on pollen tube growth may be lost if the resulting pollen is shipped or stored prior to use.

And do you really want to mess with GMO tobacco?

Bob

This is within similar doses that were used to develop Delhi 76, except in this case, only half the dna would be irradiated, reducing the number of mutations that they observed in the Delhi 76 development. https://www.google.ca/url?sa=t&sour...ID-MQFgglMAA&usg=AOvVaw0g73JGLMCMXrXpeyEAhSXe
My rough calculations, earlier was that it would require 20 minutes with 80ci of Ir192 @ 2" distance. Simple enough.
I could try it. I am curious.

Edit: food irradiation/sterilization doses are in the neighborhood of 25kGy = 2.5Mrad. So to kill bacteria, they are using 300 to 500 times the dose that I'd be giving the pollen. Also, Co60 has an energy level 3x that of Ir192.
 

Tutu

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An interesting theme is that the Oriental varieties tend to sport shorter pistils than the others. I'll have to locate additional photos, or do more observations next season, to determine if that is true.

Looking at the pictures you're posting it does indeed seem that there is a variation in pistil length. However, I wonder how much of that is simply the flower being taller in some varieties than in others. Difficult to tell this way, would have to measure that. Any way, I've got some Bursa seedlings, so hopefully their pistil size would be slightly on the short side

Bob and Tutu I have this PDF-file

This a very interesting article. My German is alright, and I have the added advantage that Google translates German better into Dutch than English.

The first thing that interested me is that the authors say that the floral features are the most reliable when it comes to variation within varieties. That is, it is the least influenced by its environment. That would mean I really want to grow Balikesir! The flowers are also the most reliable source to determine how close two varieties are to one another.

They also say that there are quite some differences to the ratio of the flowers. The length of the chalice for instance, the green part that holds the flower, can differ between varieties to quite some extent. The shape of the petals can be different too. That might indicate that the stylus may be of different lengths, but it doesn't explicitly mention it. The diffence in ratio might also contribute to the optical illusion of the pistil differing in size, I suppose.

But when it goes into describing different varieties it does say that flowers of some varieties are taller than others. That would indicate that the stylus of some varieties may be shorter than others.

All in all I would say it raises my hopes that some N. tabacum varieties may be easier fertilized by N. rustica than others. Thanks to Bob for pointing out this potential variation in the first place. If anyone still has lowers on their plants, pictures of pistils are very welcome. You might even want to try and compare flowers of different varieties!
 

deluxestogie

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As a tangent to this thread, the BBC has an excellent article on the quest for robusta coffee's resistance genes (to coffee rust fungus) in a natural robusta x arabica hybrid variety discovered in Timor. While robusta will not normally cross with arabica, the Timor hybrid will. The fungus is trying to wipe out arabica coffee production in Colombia.

http://www.bbc.com/future/story/20171106-the-disease-that-could-change-how-we-drink-coffee

Bob
 

ChinaVoodoo

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As a tangent to this thread, the BBC has an excellent article on the quest for robusta coffee's resistance genes (to coffee rust fungus) in a natural robusta x arabica hybrid variety discovered in Timor. While robusta will not normally cross with arabica, the Timor hybrid will. The fungus is trying to wipe out arabica coffee production in Colombia.

http://www.bbc.com/future/story/20171106-the-disease-that-could-change-how-we-drink-coffee

Bob

Great article. Thanks, Bob.
 

Tutu

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As a tangent to this thread, the BBC has an excellent article on the quest for robusta coffee's resistance genes (to coffee rust fungus) in a natural robusta x arabica hybrid variety discovered in Timor. While robusta will not normally cross with arabica, the Timor hybrid will. The fungus is trying to wipe out arabica coffee production in Colombia.

Very good article to say the least. Interesting by itself, because it's written well and the topic is something we growers can relate to. But indeed tangent to this thread, as it suggests that with interspecies hybridizing there may be some varieties better fit than others. Very nice how you dug this up. I suppose it would be only too much of a coincidence if my varieties found on Timor would do the trick.


I thought I would repost this article in this thread, while you're talking about pollen tubes, and remind you I have the ability to conduct the irradiation component of the pollen, and mail stamens back to you

One thing I learned from reading this article is that stored, aged seeds do not only loose viability, but that "in a range of crop plants ithas been shown that aged seeds produce inferior plantsand lower yields, even though the levels of their germinabilityare still relatively high.Stored seeds with reduced vigor are also less able towithstand a wide variety of environmental stresses". I had not known this or considered the possibility. For me it was germinating or not germinating. I never considered that the plants would grow less strong.

But I suppose this should be something to be discussed in a seed threat.
 

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Over my head, but this research paper has some gems in it.

http://www.plantcell.org/content/16/suppl_1/S98

Using our stigmaless transgenic tobacco plants (Goldman et al., 1994), we studied pollen–pistil interaction in this system, hoping to dissect the components required. The ablated surface of this stigmaless pistil is dry, and when tobacco pollen is used for pollination, it fails to hydrate. If humidity is provided, pollen hydrates but only a few short pollen tubes germinate. This finding indicates that the defect is only on the stigma side and not in the pollen. The addition of germination medium containing boron, calcium, and sugar improved tube germination, but penetration of the pistil tissues failed. Surprisingly, when we applied exudate collected from wild-type pistils, pollen tubes germinated and grew directly in the pistil tissues (Goldman et al., 1994). Currently, we are investigating the function of the proteins secreted in the exudate, some of which (LTP and PPAL) have been discussed above.

One question that arose from the discovery that exudate is sufficient for pollen tube growth in the style was whether exudates secreted on the stigmas of other species could produce a similar effect (Wolters-Arts et al., 1998). Figure 1 shows that exudate of petunia is as good as that of tobacco in restoring pollen tube penetration, whereas that of lily only enables pollen hydration and germination
 

deluxestogie

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There's some wild research out there. Stigmaless transgenic tobacco?

Petunia is in the same family (Solanaceae) as Nicotiana. The final paragraph of your quote seems to suggest to me that the "stigmaless" plants are simply missing an essential signaling chemical that is commonly secreted from the stigma of more than one genus of solanaceous plants.

Bob
 
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