Results of the hormone square and comparison with other data

Our hormone square showed the following development:

Auxin IBA→   Cytokinin BAP ↓	0 mg/l	0.02 mg/l	0.2   mg/l	2 mg/l
0 mg/l	1. Very long roots, no ramification, no callus, no fresh shoots	2. Very long roots but less than in nr.9, very little ramification, no callus, no fresh shoots	3. Similar to nr.4 but less rooting and a bit less callus	4. Dark, brown large callus, but many roots with ramifications
0.02 mg/l	5. Only little rooting, very little callus, no fresh shoots, but healthy	6. Little callus (also internodes), little rooting but some ramification, not too long	7. Little rooting, thin and young roots, green callus, healthy leaves	8. Callus, very compact, brown and green, little shoots in internodes, root ramification
0.2 mg/l	9. Very long roots, less than in nr.1, little ramification, no callus, no fresh shoots	10. some green callus, no roots, few little shoots growing from internodes	11. big green callus, no roots, little shoots growing from internodes	12. big green callus, no roots, little shoots growing from internodes, plantlets smaller
2 mg/l	13. some green callus, no roots, many shoots growing, plantlets smaller, stressed	14. some green callus, no roots, very many shoot growing from internodes, real proliferation, stressed	15. same as 14.	16. even larger green callus, no roots, less shoots growing from internodes

We tried to make a meaningful ranking for the roots and another one for the shoots. In order to do the ranking, we considered the qualities which would be most beneficial and which promise a good fitness if the explants were used for a transfer into in vivo conditions.

So concerning the roots, we determined that the explants already need to have some. This is essential for a quick adaption to taking up water and nutrients from the soil and quickly get good foothold. Even though the roots developed in the container can’t really be used in in vivo conditions, new ones can establish quickly if there is already some rooting. However, the explants from the containers in which the most roots had developed were almost too old. In Nr. 3, 4 and 8 there was very much rooting, but the roots were already very long and did not ramify much.

We thought it better to take plantlets in a stage of young roots, so that they will ramify a lot when cut and transferred into soil. In addition to that, direct rooting is better than when there's callus formed in between. Therefore, Nr. 5, 6 and 7 were considered as more suitable.

Concerning the shoots, the highest activity and cell division was found in high cytokinin (BAP) concentrations, where many little leaves were proliferating even from the internodes. Nevertheless, we regarded only well grown and vigorous shoots as desirable. If the internodes were very short, leaves were light green and overall the plantlets looked stressed, the ranking was low (this was the case in high concentrations of BAP). The best shoots could be found in Nr. 7 and 9.

Therefore, regarding both, roots and shoots the best hormone mix could be found in Nr. 7 with 0.02 mg/l BAP and 0.2 mg/l IBA. Also good plantlets in regard to suitability for in vivo conditions could be found in Nr. 5, 3 and 9.

The other group also found the most intensive rooting in Nr. 3, 4 and 8, yet also in Nr.12. Since this group probably didn’t define the ranking in regard to suitability for a transfer to in vivo conditions, they regarded these as the best ones.

This result is in accordance with scientific findings. Auxin is known to stimulate root growth, but rather in concentrations higher than 0.2 mg/l and not if there’s too much cytokinin.

On the other hand, the ranking of shoots looks completely different. This group counted the shoots on leaves and on stems of leaves. The best hormone combination for a good shoot formation was found to be 0.2 or 2 mg/l kinetin and 0.02 mg/l NAA. These were containers Nr. 10 and 14.

In any case, the effect of a combination of auxin and cytokinin was clearly visible in both. Some differences between the two hormone squares might also be due to the use of different plant hormones in both groups or just due to some mistakes in the preparation of the mixtures.

It was difficult to find some scientific reference, even though various studies on different combinations of auxins and cytokinins had been carried out in the past (two of them mentioned below, but which couldn’t be accessed). Since I’ve already spent too much time on this research and read too many unsuitable abstracts of papers, I will stop here. However, from what I’ve read and learned, our result shows the very expected effects. The auxin is stimulating roots, if it isn’t drowned by too high cytokinin concentrations. The cytokinin strongly stimulates cell division of the overground parts, which in our hormone square is best visible in the internodes.

References:

James, D., & Newton, B. (kein Datum). AUXIN: CYTOKININ INTERACTIONS IN THE IN VITRO MICRO-PROPAGATION OF STRAWBERRY PLANTS.

Karakullukçu, S., Agaoglu, Y., & Abak, K. (kein Datum). EFFECT OF DIFFERENT AUXIN-CYTOKININ COMBINATIONS ON THE IN VITRO PROPAGATION OF RASPBERRY CV. SCHÖNEMANN.

The development of Chrysanthemum in the mixed hormone square

On the 28^th of March, the square was prepared according to the following table:

In our example the auxin IBA and the cytokinin BAP were used  on a SP-0 medium.

Photos of the explants on the hormone square were taken on the 12^th of April, and I will now comment on the development that became apparent within this time.

What can soon be noticed is that the current stage of the plant development already shows clear differences due to different hormone concentrations and combinations.

Number 1 and 2 do not show any growth, in Nr. 3 little roots are visible, whereas Nr.4 shows clear formation of roots. This growing of roots is due to the well-known effect of auxin (here IBA).  

Number 5 and 9 also don’t show any growth, while in Nr. 13 some callus was formed. This formation of callus due to only BAP and no combination with auxin also makes sense since cytokinins stimulate cell division.

Though without any cytokinin and only little auxin nothing happens and if there is no auxin and only little cytokinin, nothing is growing either.

In Nr.8, with 2 ppm of auxin and 0.02 ppm cytokinin, not only roots but also calluses are forming. 

The combination of as much auxin but a little more cytokinin (Nr.12) resulted in callus and shoot formation.

Also Nr. 15 shows callus and shoots and looks similar to Nr. 12

Unfortunately it seems that the formation of roots and shoots cannot be achieved with only one mix of concentrations but a succession of different combinations would have to happen. If first the shoots are generated and then the rooting should happen there is the difficulty of having callus in between. Since callus between the vascular bundles of the stem and the vascular system of the roots reduces the ability to take up nutrients and water, direct rooting of the shoots is desired. 

Plant hormones applied in in vitro cultures of Diospyros kaki

Diospyros kaki (Persimmon) was regenerated by direct organogenesis from in vitro grown leaf callus. A remarkable shoot multiplication rate of 25 was achieved within 18 weeks from 4 weeks old clumps applying trans-zeatin (2 mgl-1) and indole-3-butric acid (0.2 mgl-1). 

You plan to establish a mass propagation.

Which group of growth regulators (plant hormones) do the applied plant hormones in this report belong to?

IAA (indole-3-acetic acid) and IBA (indole-3-butyric acid) are naturally occurring plant hormones of the auxin- family. IAA is the most common and best known plant hormone among the auxins. Auxins play an important role in the coordination of many growth and behavioural processes in the plant. They contribute to organ shaping and are therefore fundamental for a proper development of the plant itself.

BAP (6-benzylaminopurine) is a synthetically produced cytokinin of the adenine-type. It stimulates cell division and influences the formation of blossom and fruit.

TDZ (thidiazuron) is a cytokinin of the phenylurea-type.

The plant growth regulator t-Zea (trans-Zeatin) also belongs to the family of cytokinins and occurs naturally.  It promotes growth of lateral buds and stimulates cell division to produce bushier plants.

http://en.wikipedia.org/wiki/Indole-3-acetic_acid

http://en.wikipedia.org/wiki/Indole-3-butyric_acid

http://en.wikipedia.org/wiki/6-Benzylaminopurine

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

Why is there still a caveat about genetic authenticity of the clonal offspring?

Plants that have been produced by plant tissue cultures are normally identical clones of the mother plant. However, there is a phenomenon called somaclonal variation.

Somaclonal variation means all variation originating in cell and tissue cultures, where mistakes in the reading of genetic information happen frequently.

The main reasons for variations are chromosomal rearrangements, but the variations can be genotypic or phenotypic. In the latter case changes can be either genetic or epigenetic in origin. Typical genetic alterations are changes in chromosome numbers, chromosome structure and DNA sequence. Typical epigenetic related events are gene amplification and gene methylation.

Somaclonal variation is an important source of genetic variability. Yet for operations which require clonal uniformity it can be a great disadvantage, like in this case the rapid propagation of elite genotypes from Diospyros kaki.

Suggest how genetic authenticity is to be verified

There might be some morphological changes visible to the naked eye. If there are no changes visible, plant screening procedures can be applied to detect somaclonal variation.
There is a method called cytogenetic analysis. With this method chromosomal alteration and ploidy changes can be highlighted.  
The different molecular analytic techniques work with molecular markers to identify DNA sequences. 
  

https://en.wikipedia.org/wiki/Somaclonal_variation

http://cdn.intechopen.com/pdfs/40179/InTech-Somaclonal_variation_in_tissue_culture_a_case_study_with_olive.pdf

original article:
Sutter F. (2010), Diospyros kaki L. Plant regeneration via in vitro leaf derived callus

How could an in vitro propagation system for Alstroemeria be established using plant hormones?

Growth of in vitro cultures can be managed accurately by plant hormones. Alstroemeria is a popular cut flower grown from rhizomes. Varieties change frequently on the market and plant breeders depend on quick techniques to promote and propagate new cultivars in time. Read the paper indicated below and discuss how an in vitro propagation system might be established by using plant hormones.

The paper Micropropagation of Alstromeria x hyprida ‘Juanita’ shows the results of a study about the effects of auxins, cytokinins and growth retardant on the growth patterns of Alstroemeria. The study had been carried out in order to find a way to increase the efficiency of in vitro micropropagation of Alstroemeria.

Since Alstroemeria is grown from rhizomes, the aim was to obtain a high amount of branched and easy-to-root rhizomes containing numerous, short generative shoots. The experiments with different concentrations of auxins or cytokinins alone or combined with a growth retardant should demonstrate, from which method these quality characteristics will result.

The results show, that an application of the cytokine BAP at a high concentration is not advisable because the rooting ability of the rhizomes becomes poor. Also the combination of BAP at a low concentration with kinetin or 2iP did not stand the test. However, a low concentration of BAP together with paclobutrazol or flurprimidol resulted in a high number of aerial shoots, the reduction of their length and a high rooting ability of the rhizomes.

Therefore, (according to Fig. 2) this is the application that could be suggested: 1.5 mg/l BAP + 0.1 mg/l flurprimidol and a basic medium (BM).

If the auxin NAA was added to the medium, an application of paclobutrazol or flurprimidol had a positive effect on the number of roots per rhizome and suppressed areal shoot and root elongation. This effect was though reversed, if BAP was added in addition to one of the growth retardants (paclobutrazol or flurprimidol).

So this is a possibility to stimulate rhizome rooting while inhibiting the root and areal shoot elongation: Medium containing 1mg/l NAA + 0.1 mg/l paclobutrazol.

Nevertheless, the results do not show here, whether this combination increases the number of aerial shoots per rhizome. So if there is a need to increase aerial shoot growth, the first possibility should be chosen. 

Original paper:
Micropropagation of Alstromeria Hybrida ‘Juanita’: http://www.actahort.org/members/showpdf?booknrarnr=447_32