The results and other sciencey stuff!!
So, I have found out quiet a lot of stuff about these seeds. As I have told you before both species were of different maturity. One lot were collected in 2006 and one lot 2010. The more mature the seed, the more difficult they are to grow. But! The seeds that did better were Dactylorhiza fuchsii, surprisingly, these were the more mature seed out of the two did better and at first I didn't know why but after a lot of reading and I mean a lot! I found out that D-glucose, one really good branch of glucose, really encourages seeds to grow well in asymbiotic germination. After more reading, it is possible that combining sucrose and D-glucose might help for better germination, fructose on the other hand isn't worth considering because one of the media F522 had fructose and sucrose in it and it didn't seem to work very well with either species.
From the experiment, the two that are best to use are the T849 and the MS, mainly because they both contained nitrogen and sucrose. The T849 was slightly better because of the D-glucose in it so basically, in the future, if I decide to do a MSc, I can find out if different sugar levels or the combination of carbohydrates and sugars is better for the orchids or not.
It was also very cool to find out that if you have the right components and nutrients in a media, you don't even need a fungus to germinate the seeds! All in all it was a good experiment, the figures are below Orange is T849, Pink is MS and Green is F522. I won't reveal too much but have a look and see what you think of what you can see, but it is clear to see that there is better germination in the T849 and MS. The red bits represents mould that started infecting the petri dishes, which is annoying but, all in all I am happy with the experiment!!
From the experiment, the two that are best to use are the T849 and the MS, mainly because they both contained nitrogen and sucrose. The T849 was slightly better because of the D-glucose in it so basically, in the future, if I decide to do a MSc, I can find out if different sugar levels or the combination of carbohydrates and sugars is better for the orchids or not.
It was also very cool to find out that if you have the right components and nutrients in a media, you don't even need a fungus to germinate the seeds! All in all it was a good experiment, the figures are below Orange is T849, Pink is MS and Green is F522. I won't reveal too much but have a look and see what you think of what you can see, but it is clear to see that there is better germination in the T849 and MS. The red bits represents mould that started infecting the petri dishes, which is annoying but, all in all I am happy with the experiment!!
The discussiony part of the experiment, basically what I think has happened!
The main results from the research, when investigating the germination rates on the three types of media showed that the germination rates were considerably higher in one of the two species of Orchid on the species specific media. In an experiment designed for this kind of comparison, human error or certain features of the experiment may have limited the results. It was easy to define that the T849 media is the better choice for asymbiotic germination. The MS proved to be adequate for immature seed, however, the nutrient content and the mono and disaccharide content may need to be changed to make it more competent for Orchid germination. The medium that failed was the F522 and this was possibly due to the nutrient content found within. The differences between the species showed significant difference but it is clear to see from the results section that there were visible differences between media but only a natural difference occurred statistically. The experiment was terminated at week 33 when one of the two species showed emergence of shoots (leaves) and also because many of the petri dishes were drying out or contaminated with mould, started to appear at week 10 after initial sowing. Although all petri-dishes were sealed with the aid of parafilm, the climatic conditions in which they were stored may have caused the parafilm to dry and split allowing for pathogens to enter the culture plates and infect the media. Studies undertaken by Seaton and Hailes (1989) allowed for the moisture content in asymbiotic germination to be an important factor for Orchid propagation. It has been found that moisture and temperature may affect the pathogens found in petri dishes. The moisture in the petri dishes may have encouraged the growth of mould and bacteria in the culture plates, causing the data to be unavailable for some weeks of the experiment (week 10 on MS medium and 18 for T849). The appropriate moisture for Orchid propagation has been found to be 5.6% humidity at a temperature of 20°C. The seeds were initially stored at a temperature of 20°C but when moved into the analytical laboratory for storage in the light, the temperatures may have varied. Unfortunately for the experiment, the temperature was not monitored and it may have been for this reason that the mould and bacteria inhibited the media (Pritchard, 1989). The moisture content found within the petri dishes used may have enhanced the progression of protocorm development in the seeds. It allows the media to continue releasing the nutrients and it also allows the seed sown onto different media to be more viable during the development stages (Seaton and Hailes, 1989). The nature of the mould and bacteria is unknown but it is possible that they were already present in the room in which the seeds were stored. There is also the possibility that the infections came from the moment when the seeds were sown. Even though the seeds were disinfected and sown in sterile conditions using the “packet method” recommended by Seaton and Ramsay (2005), it seems not all pathogens were eliminated at the moment of disinfecting. Household bleach (10%) was diluted in sterile water but from the problems caused by the mould, it is suggested that this may have not been an adequate disinfection process. A 50% glycerol and 60% ammonium sulphate solution has been found by Chang et al. (2010) to be more appropriate to remove bacteria and fungus such as Streptomyces (which is a form of prokaryote) and also some branches of hyphae type fungi. The mould found in the petri dishes is thought to be penicillin mould which has the same type of structure as Streptomyces. Although a bleach cleansing was adequate for the first three months of the experiment, it is only viable if culture plates are stored at temperatures lower than 20°C (Seaton and Ramsay 2005).
The experiment has now been terminated and the seeds will soon be placed in the dark for an additional three months as they require two dark treatments the first year after initial sowing. This will be done in July as this is when the seeds were first sown. Dactylorhiza should normally be sown in October as the seed development usually occurs over the winter and the summer but because of the nature of the experiment and the limited time given for the project the sowing had to be moved forward by 4 months to allow for the data to be collected correctly.
The experiment has now been terminated and the seeds will soon be placed in the dark for an additional three months as they require two dark treatments the first year after initial sowing. This will be done in July as this is when the seeds were first sown. Dactylorhiza should normally be sown in October as the seed development usually occurs over the winter and the summer but because of the nature of the experiment and the limited time given for the project the sowing had to be moved forward by 4 months to allow for the data to be collected correctly.
A little conclusion!
From the results it is possible to measure the outcome after the 33 weeks of the project. It is best to use a media that is similar to the T849 medium that was used in the experiment as components such as D-glucose encourage the growth and development of seeds but also it has been specifically designed for terrestrial Orchid species. It is advisable for Orchid growers to add glucose as well as sucrose to a media similar to the MS medium used for the experiment as the experiment shows that glucose is the monosaccharide that helps encourage the germination. From the research, it seems possible that adding sucrose, fructose and other mono or disaccharides would improve the germination of hardy Orchids, then this would be the preferred method, in order for this hypothesis to be true. Further research on the effects of combining two types of carbohydrate is advisable.
From this experiment it is clear that D-glucose is the preferred element to add to an asymbiotic mix (Ponert et al. 2013), but here it is clear to see that the maturity has not affected the growth of D. fuchsii. If there are limited time periods in which to grow seed for commercial production, addition of D-glucose or the combination of other mono or di-saccharides is suggested on asymbiotic germination. Researching further techniques methods of germination need to be considered carefully to ensure that the seeds are not inhibited or damaged by the high nutrient content that may or may not be present in the media and to allow for seeds of two types of maturity to successfully germinate.
Using another method of disinfecting the seed than the one used, would aid the germination process and the sterility of the work, additional research is desirable as the techniques suggested by Chang et al. (2010) are proven to be adequate for asymbiotic germination due to the effectiveness of eliminating all pathogens with the use of the glycerol and ammonium sulphate; Although the main aim of the experiment was not to determine whether purchasing the pre-prepared media would be advantageous for horticultural and commercial growers; it may well lead to a reduction in production costs as it requires less time spent in preparation and less expenditure in the purchase of less individual components. Further research on the economical values that using a pre-prepared media may bring to an Orchid production site is recommended, especially for such rare species as the ones seen here. This recommendation would be of value to establishments or organisations dedicated to Orchid Conservation across the UK (Richard et al. 2007).
The aims of the experiment were met by proving which media has done better for asymbiotic germination, the natural variation allows us to see that the media to choose is the T849 as the correct nutrients D-glucose were present to activate the growth and development of the protocorm, the preferable species to use is D. fuchsii as there is obviously a positive reaction to asymbiotic media. It is possible to determine that D. purpurella is not and adequate species as they may prefer a symbiotic germination process to encourage nutrient exchange.
From this experiment it is clear that D-glucose is the preferred element to add to an asymbiotic mix (Ponert et al. 2013), but here it is clear to see that the maturity has not affected the growth of D. fuchsii. If there are limited time periods in which to grow seed for commercial production, addition of D-glucose or the combination of other mono or di-saccharides is suggested on asymbiotic germination. Researching further techniques methods of germination need to be considered carefully to ensure that the seeds are not inhibited or damaged by the high nutrient content that may or may not be present in the media and to allow for seeds of two types of maturity to successfully germinate.
Using another method of disinfecting the seed than the one used, would aid the germination process and the sterility of the work, additional research is desirable as the techniques suggested by Chang et al. (2010) are proven to be adequate for asymbiotic germination due to the effectiveness of eliminating all pathogens with the use of the glycerol and ammonium sulphate; Although the main aim of the experiment was not to determine whether purchasing the pre-prepared media would be advantageous for horticultural and commercial growers; it may well lead to a reduction in production costs as it requires less time spent in preparation and less expenditure in the purchase of less individual components. Further research on the economical values that using a pre-prepared media may bring to an Orchid production site is recommended, especially for such rare species as the ones seen here. This recommendation would be of value to establishments or organisations dedicated to Orchid Conservation across the UK (Richard et al. 2007).
The aims of the experiment were met by proving which media has done better for asymbiotic germination, the natural variation allows us to see that the media to choose is the T849 as the correct nutrients D-glucose were present to activate the growth and development of the protocorm, the preferable species to use is D. fuchsii as there is obviously a positive reaction to asymbiotic media. It is possible to determine that D. purpurella is not and adequate species as they may prefer a symbiotic germination process to encourage nutrient exchange.