Many people have heard about two seemingly disparate concepts: recalcitrant (or difficult to grow) plants and liquid chromatography. Plant tissue culture or in vitro culture is a valuable technique which allows for the growth, multiplication, and manipulation of plants in sterile and controlled culture conditions and which requires only very little starting material. Plants are grown in a growth medium, usually agar based, and are supplemented with carbon, micro- and macro-nutrients to promote and support growth.
Medium in which they are grown is often also supplemented with various plant growth regulating chemicals, termed plant growth regulators or phytohormones, which can be employed to direct plants towards specific growth patterns. The most commonly used classes are auxins, the first PGR discovered, and cytokinins. The balance of these two regulators was determined to be pivotal in directing plant growth and was described by Skoog and Miller in cultures of tobacco leaves in 1957. Simply stated, high auxin and low cytokinin lead to root production; low auxin and high cytokinin to shoot production; and a balance of the two leads to the production of a mass of undifferentiated cells, a type of tissue referred to as callus.
Plant tissue culture is based on the concept of totipotency, that is the phenomenon in which any single plant cell has the capacity to de-differentiate and re-differentiate into any other type of cells and eventually the whole plantlets. For this reason, thousands or even millions of plants can be propagated from a single leaf from a parent plant, deemed an explant. In comparison with traditional propagation methods which may require taking a large branch or root cuttings, this makes tissue culture an attractive technique for propagating plants for which limited material is available. Endangered plants are one such group, as the production of more of these plants is obviously desirable, however, populations have been reduced to small numbers with some even being represented by a single individual, making the collection of large amounts of tissues unfeasible, particularly for smaller individuals.
Unfortunately, not all plants abide by the principles originally established by Skoog and Miller, and disproportionately medicinal and endangered plants, tend not to easily follow these principles compared to other species. Though perhaps unexpected at first glance, this is logical as these species often have specialized characteristics or growth requirements which lead them to be endangered and/or medicinal in the first place. Conventional methods use an approach to solve this problem which involves attempting to grow the explants on diverse combinations of micro- and macro-nutrients coupled with combinations of different classes of plant growth regulators, the main ones being: auxins, cytokinins, gibberellins, jasmonates, and salicylates.
Labs may also employ diverse inhibitors in an attempt to modify the endogenous levels of these hormones in tissue as plants which naturally have already high auxin levels, for example, may be more resistant to the production of shoots, or may be more sensitive to the addition of supplemental auxin. The complexity of plant chemistry means that this can be a time consuming and ultimately expensive fruitless effort, with researchers essentially guessing at what may overcome the recalcitrance.
Meaning literally colour (“chrom”) writing (“graphy”) the average person’s experience with chromatography may extend only to simple experiments encountered in grade school where a black marker is separated to see the rainbow of colours that comprise this ink on a coffee filter, or to a passing reference on popular crime investigation show’s such as CSI, or forensic files. The technique, however, has been in use in research and commercial labs for decades, and though it is most often associated with chemists the technique was first defined by the botanist Mikhail Tsvet in the late 1800’s with the goal of separating the plant photosynthetic pigments: xanthophylls (yellow), carotenoids (orange) and chlorophyll (green). This technique involves separation of compounds from a mixture across a column generally packed with silica and separated compounds then enter into a detector which is capable of measuring and identifying these compounds.
Propagating Recalcitrant Plants
In our recent study, we have brought together these two concepts to propose a novel approach to the problem of propagating recalcitrant plants, by first quantifying the levels of endogenous plant growth regulators and using this as the starting point for propagation efforts. To do this we have developed a simple, efficient and low-cost method for the analysis of these compounds. This method uses relatively inexpensive instrumentation, easily disposed of solvents and does not require extensive chemistry background, all of which represent major obstacles for plant tissue culture labs which may wish to implement these technologies.
Plant tissues are first flash frozen, ground and extracted in 50 % acidified methanol and extracted via sonication. After being filtered and diluted the samples are ready for analysis. This method employs liquid chromatography with detection by mass spectrometry to quantify in a single 5 minute run eight major plant growth regulators including three cytokinins – zeatin, benzylaminopurine (BA), 6-(γ,γ- dimethylallylamino)purine (2-iP), auxin (indole-3-acetic acid; IAA), gibberellic acid (GA3), abscisic acid (ABA), jasmonic acid (JA), and salicylic acid (SA), as well as the precursor of JA: 12-oxophytodienoic acid (OPDA) and the active form of JA: jasmonic acid isoleucine (JA-Ile) as well as the salicylic acid derivative acetylsalicyclic acid (ASA), commonly known as aspirin. Samples are then separated by liquid chromatography using a gradient method with mobile phase constituents: 10 mM ammonium acetate, pH 9.0 and 100 % methanol.
Analytes are detected using single ion recording which allows for enhanced selectivity and good sensitivity. This method was validated through repeated injections across multiple days and preparations of extracts and across eight plants species which included medicinal plants (sweet wormwood, fennel, and St. John’s wort) commercial species (potato and banana), ornamentals (African violet) and trees (American elm) and three tissue types (shoot, root and seed). It was found to have excellent accuracy, precision and reproducibility making it a method that should be easy to reproduce in other labs.
Additionally, inclusion of the three derivative compounds (JA-Ile, OPDA and ASA) demonstrate that in addition to being useful in measurement of the compounds already included in the method, it can also be used as a starting point for the addition of further related compounds, in labs for which the method is not already comprehensive.
This method, therefore, provides the technical method required for a new approach to overcoming recalcitrance in diverse plant species, which does not require expensive or especially hazardous chemicals, and which may be employed across labs with a range of technical experience.
These findings are described in the article entitled A simple and efficient method for analysis of plant growth regulators: new tool in the chest to combat recalcitrance in plant tissue culture, published in the journal Plant Cell, Tissue and Organ Culture. This work was led by Lauren A E Erland & Praveen K Saxena from the University of Guelph.