4.5 Inoculation techniques of plants

Blog entry week 16-20/2015

 Somaclonal variation could not be reported within different samples of callus derived from one plant of origin. Why is that an important fact considering a clonal mass propagation of neem?

 Tissue culture-induced genetic variations i.e., somaclonal variations have been reported to occur frequently in clonally propagated plants (Larkin and Scowcroft, 1981).

Although neem produces a large number of seeds per tree, it is difficult to store the seeds for extended periods of time (Sacandé and Hoekstra, 2003). Moreover, there are another studies, that proves that vegetative propagation of neem using conventional methods is not possible (Narayan et al., 1985). This gives more scope for potential applications of in vitro culture techniques towards neem improvement.

 There are also studies, that demonstrated the possibility for mass clonal propagation of a 50-year-old neem tree by nodal segment cultures. MS medium supplemented with BAP (1 M) and CH (250 mg l⁻¹) medium was used for recurrent shoot multiplication at a rate of 7–8-fold every 5 week and this rate of shoot multiplication was maintained for almost 5 years. The shoots could be readily rooted with a frequency as high as 82%. Transplantation survival of these plants was more than 87% (Chaturvedi et al., 2004).

 Why was callus used to perform this investigation?

 Simple Sequence Repeat (SSR) markers in that research were used to confirm the genetic stability of callus. These studies focuses on standardizing conditions for establishing callus cultures using novel explants from a mature neem tree and test genetic stability of the calli using molecular markers (Ramamurthy et al., 2012).

Which conclusions are possible based on the fact, that callus derived from one stock plant remains genetically stable but compared with two other neem plants shows some polymorphism?

 PCR amplifications carried out using highly polymorphic microsatellite markers have revealed that the genomic DNA at specific loci (Ai_4, Ai_11, Ai_13 and Ai_34) from various explant derived calli do not exhibit any significant polymorphism in comparison to the mother plant, confirming their genetic stability (Ramamurthy et al., 2012).

Literature:

1. Larkin, P.J. and Scowcroft, W.R. (1981). Somaclonal variations - a novel source of variability from cell cultures for plant improvement. Theor. Appl. Genet. 60:197-214.

2. Sacandé, M. and Hoekstra, F.A. (2003). Optimising conditions for neem (Azadirachta indica) seed longevity. p.761-773 In: R.D. Smith, J.B. Dickie, S.H. Linington, H.W. Pritchard and R.J. Probert (eds.), Seed Conservation: Turning Science into Practice. Kew: Royal Botanic Gardens, Kew.

3. Narayan, P. and Jaiswal, V.S. (1985). Plantlet regeneration from leaflet callus of Azadirachta indica A. Juss, J. Tree Sciences 4:65-68.

4. Chaturvedi, R., Razdan, M.K. and Bhojwani, S.S. (2004). In vitro clonal propagation of an adult tree of neem (Azadirachta indica A. Juss.) by forced axillary branching. Plant Science 166:501-506.

5. Ramamurthy A., Kag B., Hegde V., Malarini Loganathan R., Saiyed T., Sathyanarayana B.N.,  Gowda M. (2012) Studies on In Vitro Regeneration from Various Explants of a Mature Neem (Azadirachta indica) Tree. ISHS Acta Horticulturae 961: VII International Symposium on In Vitro Culture and Horticultural Breeding. p. 449-456.

Blog entry week 13 - 15

Botanists versus molecular biologists

It is time to summarize the advantages and disadvantages of these two methods and, what is important – limits of these both methods. My answers:

1. What are the advantages and disadvantages of the classical botanical method to describe plant species?

Advantage definitely is fact, that method is cheap and fast – person with knowledge in botanic always can identify plant in it natural area, in the field without collecting samples, damaging natural area (often we need more than one sample) working in the lab.

Disadvantages. Do not forget that Carl Linnaeus lived 300 years ago and during this time any scientific discipline might change and update itself. For example: Echinacea is the genus, but there are nine different species.

Two of the most popular species of Echinacea are angustifolia and purpurea. The botanical names are Echinacea angustifolia and Echinacea purpurea.

The purpose of assigning botanical names to plants is so that each plant has a single name that’s used worldwide to identify it. Botanical names prevent people from confusing different species of plants, but also indicate that different species are related (Advantages and Disadvantages of Common Names & Botanical Name, https://ehomoeopathy.wordpress.com/2011/10/21/advantages-and-disadvantages-of-common-names-botanical-name/), so it means that we definitely need deep knowledges even to identify sample within genus.

2. What are advantages and disadvantages of barcoding?

Advantages: DNA barcoding is a fast, accurate, and standardized method for species level identification, by using short DNA sequences (Lebonah et al., 2014). DNA barcoding is the standardized research that facilitates biodiversity studies like species identification and discovery. This technique helps researchers to understand genetic and evolutionary relationships by assembling molecular, morphological, and distributional data (Savolainen et al., 2014). Additional advantages of the method would be the possibility of identifying individuals at any stage of development, and the prospect of discriminating between morphologically identical species (DNA barcoding and…, 2010), that is not possible, using classical bothanic.

Disadvantages: On the first view, disadvantages does not exist. But I still think - What about hybrids? Do we have necessary primers for them? And there are possibility of simple human mistake in the lab and – results of identification of exact sample of plant are not reliable anymore, but in orchard you will always recognize necessary sample and not make a mistake (of course, if you have a knowledges).

3. Give an example of plant species where barcoding led to the revision of the description of plant species.

That example is not so easy to find, but there were researches about identification of plant species in honey, also honey quality (Bruni et al., 2014), that is not possible without barcoding.

4. In your opinion, what is the best strategy to do plant taxonomy? Would you rather trust the botanist or the molecular biologist?

 If question is only - would you rather trust the botanist or the molecular biologist, I definitely would trust molecular biologist, because systematic of botanic constantly changes, experts every year discover new and new species and often even biggest experts argue about belonging of exact species or necessary of new species or subspecies. But for reliable plant identification, taxonomy is too early to refuse from taxonomists at all. Even high quality lab and experts can not replace persons, who know plants in the orchards and for the high quality plant taxonomy we need to have synergy, not competition between molecular biology and taxonomists.

Literature:

D. E. Lebonah, A. Dileep, K. Chandrasekhar, S. Sreevani, B. Sreedevi, and J. Pramoda Kumari (2014) DNA Barcoding on Bacteria: A Review. Volume 2014 (2014), Article ID 541787, 9 pages.

V. Savolainen, R. S. Cowan, A. P. Vogler, G. K. Roderick, and R. Lane, “Towards writing the encyclopaedia of life: an introduction to DNA barcoding,” Philosophical Transactions of the Royal Society B: Biological Sciences, vol. 360, no. 1462, pp. 1805–1811, 2005.

DNA barcoding and traditional taxonomy unified through Integrative Taxonomy: a view that challenges the debate questioning both methodologies. In: Biota Neotrop. vol.10 no.2 Campinas Apr./June 2010.

I. Bruni, A. Galimberti, L. Caridi, D. Scaccabarozzi, F. De Mattia, M. Casiraghi, M. Labra (2014) A DNA barcoding approach to identify plant species in multiflower honey. Food Chemistry 170 (2015) 308–315.