Data from my project compared with other data

ROOTS Ranking (Groupe A)
NAA→ ; Kinetin ↓	0ml	0.03ml	0.3ml	3ml
0ml	4	4	8	9
0.03ml	3	3	7	8
0.3ml	2	2	7	8
3ml	1	2	5	7

ROOTS Ranking (Group B)
IBA→ ; BAP ↓	0ml	0.03ml	0.3ml	3ml
0ml	6	6	9	9
0.03ml	7	7	7	9
0.3ml	6	3	3	3
3ml	3	3	3	3

Group A worked with Saintpaulia sp. and the hormones NAA und Kinetin and

Group B worked with Chrysanthemum plants and the hormones IBA and BAP.

The tables show the different combination of hormones and the effects on the root and callus formation. We gave the root/callus formation a ranking from 1 (worst) to 9 (best) with regard to further propagation. A 9 would be many short, sick roots and a 1 would be brown tumour without any roots.

The results of the two groups where quite different. It is also hard to compare the two cultures, because Saintpaulia sp. forms rosettes and Chrysanthemum grows tall with long shoots.

In general we can say that it is better to put more auxin than cytokinine. The best results in both hormone squares where the ones with 3ml auxin and 0ml cytokinine (box number 4, compare box numbers with previous blog). Both groups had the worst results with 3ml cytocinine and no auxin added. Everything between is a bit of a mix of rankings from 1 to 9. However, this is just with regard to the rooting. 

On boundless (2013) I found an article about what we should expect with different auxin and cytokinine combinations: "When cytokinin and auxin concentrations are approximately equal in a plant, plant cells do not differentiate. If only auxin is present, plant cells will enlarge, but will not divide. If only cytokinins are present, no changes occur in the plant cells. If cytokinins are present at higher concentrations than auxin, both cell enlargement and division will occur. Increase in auxin levels causes root formation."

Most of these effects are already visible on our cultures and the others might just need a little more time. On the other hand, we had already effects of just cytokinine added and no auxin.

References: 

BOUNDLESS. (2013). Cytokinins Control Cell Growth and Differentiation
. Abgerufen am 06. Mai 2013: www.boundless.com

My own hormone square

The members of my groupe made a mixed hormone square with shoot tips and stems of Chrysanthemum. We used different amounts of auxin (IBA) and cytokinin (BAP) in 16 plastic boxes to see what effects different hormone combinations can have.

Auxin (IBA) is known to cause root and callus formation by high concentration and shoot formation by low concentration.

Cytokinin (BAP) is more or less the other way around.  It causes shoot formation by high concentration and root or callus formation by low concentration.

IBA→ ; BAP ↓	0 ml	0.03 ml	0.3 ml	3 ml
0 ml	1	2	3	4
0.03 ml	5	6	7	8
0.3 ml	9	10	11	12
3 ml	13	14	15	16

Nr. 1 : I could not get a good picture from of this box because of the condensation on the inside of the lid. That is why I took a picture of the bottom of the box. The white lines are the thin roots in the medium. We added neither auxin nor cytokinin in this box.  Although we did not add any hormones, the plant cuttings built roots. There are two possibilities for this; the plant cuttings could produce the hormones needed for direct root formation themselves or we did not work enough systematic and added hormones into the wrong media.

Nr. 4 : To this box we added 3ml of auxin and no cytokinin. Now there are lots of shoots and they are thicker and shorter than in box Nr.1. 

In the second row the plants already made little calluses and in box Nr.8 there are callus and roots at the same time.

In the third row the plant parts show callus formation and no roots. Nr. 9 just shows very little callus compared to the other boxes of this row.

Nr. 13: We added no auxin and 3ml of cytokinin in this box. There is now a lot of callus visible.

Nr. 16: We added 3ml of auxin and cytokinin to the media of this box. The plant parts show lots of callus.

In general, I can say that the expected effects of IBA and BAP are also true for my own experiment. In the first row (Nr. 1 to 4) occurred no callus formation. The further down the rows or the more BAP added to the media, the more callus was built.

In addition, there are more leaves the further to the right or the more IBA added in the square. I can’t tell if that is just because of the hormones we added or because the plant parts didn’t have all the same quantity when we transplanted them into the hormone square.

plant hormones and genotypic variation in Diospyros kaki in vitro culture

In the scientific paper ‘Diospyros kaki L. Plant regeneration via in vitro leaf derived callus’ (F. Sutter, 2009) an experiment with the aim to figure out the suitability of Diospyros kaki to a milder climate is described.

For this experiment a combination of the following plant hormones is used.

Trans-Zeantin, a naturally occurring cytokine, is induced to simulate cell division and shoot formation.

Thidiazuron is neither an auxin nor a cytokinin.  “TDZ (Thidiazuron) has shown both auxin and cytokinin like effects, although chemically it is totally different from commonly used auxins and cytokinins.” (African Journal of Biotechnology, B. Guo et al., 2011)

IAA (indole-3-acetic acid), a major auxin involved  in many physiological processes of plants.

IBA (indole-3-butyric acid) is an auxin used to promote the formation of roots.

Apart from genotypic variation the replication of Diosparos kaki via in vitro leaf derived callus was successful.  Genetic variation in callus cultures due to mutation occurs often and is called somaclonal variation.

Somaclonal variation has positive and negative effects. It mainly depends on the aim of the wok what the advantages and disadvantages are. If your target is to produce as many identical plants of one individual with ideal qualities, somaclonal variation is not wished and have a major economic impact on this industrie. On the other hand, somaclonal variation is wished when the aim is to produce plants with new quantities like disease resistance, improvement of nutritional quality, adaptation of plants to stress conditions, resistance to herbicides etc.

Sutter mentioned that further trials with frost tolerant and early maturing cultivars are planned. Somaclonal variation could help to achieve this qualities.

Some facts for somaclonal variation:

- Somaclonal variations may show novel mutations.

- Somaclonal variations may reduce time required for mutation breeding.

- Many somaclonal variants show undesirable features such as reduced fertility, growth rate etc.

- The variation is not always heritable

- Selected clones show unpredictable and uncontrollable variants.

For detecting the variants, computer based automated cell sorting devices can be used to screen cells and separate variant cells automatically. These variant cells can then be destroyed or further regenerated.  This information I have found on http://concerncrisis.blogspot.ch/2009/05/somaclonal-variation.html without any further information about this method. So I don’t know how or if the computer based analysis are practiced in in vitro cultures.

ISSR (Inter-simple sequence repeat) and RAPD-PCR (polymerase chain reaction) are useful techniques for detection of somaclonal variation.

Some changes in the genetic code show visible effects and can easily be detected, others don’t have any effects at all.

In several experiments, scientists bring appearance of somaclonal variation in relation with callus age, hormone application and UV-C radiation. This information could help to decrease or increase genetic variation in in vitro tissue cultures.

Thidiazuron (African Journal of Biotechnology, B. Guo et al., 2011)

http://www.academicjournals.org/ajb/PDF/pdf2011/17Aug/Guo%20et%20al.pdf

Plant hormones to buy with brief information about their effects

www.toku-e.com

General plant hormone information

www.plant-hormones.info

Methoed to detect variants and reasons when it occurs more often

www.genetic-id.com

www.dnalc.org/resources/animations/gelelectrophoresis

http://concerncrisis.blogspot.ch/2009/05/somaclonal-variation.html

http://www.bio21.bas.bg/ipp/gapbfiles/v-33/07_1-2_03-11.pdf

Effects of plant hormones

Plant hormones play a vital role in regulating growth and development. There are many different types of hormones and their effects depend on several factors like plant type, stage of the plant’s development, amount of hormones and combination of hormones. Most hormones have more than one function eg. the following are responses that auxin is known to cause:

• Stimulates cell elongation 

• Stimulates cell division in the cambium and, in combination with cytokinins in tissue culture 

• Stimulates differentiation of phloem and xylem 

• Stimulates root initiation on stem cuttings and lateral root development in tissue culture 

• Mediates the tropistic response of bending in response to gravity and light 

• The auxin supply from the apical bud suppresses growth of lateral buds 

• Delays leaf senescence 

• Can inhibit or promote (via ethylene stimulation) leaf and fruit abscission 

• Can induce fruit setting and growth in some plants 

• Involved in assimilate movement toward auxin possibly by an effect on phloem transport 

• Delays fruit ripening 

• Promotes flowering in Bromeliads 

• Stimulates growth of flower parts 

• Promotes (via ethylene production) femaleness in dioecious flowers 

• Stimulates the production of ethylene at high concentrations

(Refference: http://www.plant-hormones.info/auxins.htm)

In the observed article (Micropropagation of Alstroemeria x hybrida `juanita‘ ) the effects of different kind of cytokinines and auxines alone or in combination with growth retardant were studied in relation to rhizome branching, arial shoot production and rooting of rhizome.

Alstroemeria x hybrida is a plant which can be reproduced by rhizome. According to that, large numbers of roots per rhizome and many strong shoots is the aim of in vitro grown Alstroemeria x hybrid cultures.

The results of this experiment show that one hormone has more than one effect and in combination with one or several other hormones the results are again different. BAP, a cytokinin, has an effect on shoot length and number of roots per rhizome. A smaller amount of BAP causes greater shoot length and greater numbers of roots per rhizome. Combined with paclobutrazol and flurprimidol, two growth retardants, the shoot length is shorter than with just BAP. When also added NAA, an auxin, to the media the number of roots per rhizome increases rapidly.

According to these results, the best effects are achieved with little BAP (1.5mg/l) combined with NAA and flurprimidol.

Plant hormones are also used as herbicide, which means you have to know what you are doing by adding hormones to a culture. The right amount at the right time is vital for a plant. In nature the plants find the right amount of hormones at the right time themselves. In an in vitro culture the hormone application has to be well studied to success.  Hormones are just needed in a very small amount, in in vitro cultures that can be hard to achieve. In practice there is often a big amount of the whole matrix mixture made so that the amount of hormones in every little plant box is accurate.

Overall, plant hormones can have many different effects and for in vitro cultures the challenge is to get the time, the amount, the type of hormone and the combination of hormones right.