Tools to Improve the Breeding Efficiency

Breeding a new cultivar needs 5–15 years according to the species and the breeding objectives from bioprospecting up to cultivar registration. This is a very long time for companies developing and trading plant based products. To react more quickly to the requirements of the stakeholders, methods to accelerate the breeding pro-cedures must be taken into account. One of the possibilities is the acceleration of generation succession by vernalisation and cultivation in the greenhouse allowing to increase the number of generations per year (Pank and Schwarz2005). Other approaches are the use of morphological, phytochemical and genetic markers at a very early stage in the reproduction cycle, as well as rapid and cheap measurement Fig. 6.1 In-vitro

preservation and reproduction of the most interesting genotypes of Artemisia annua L

methods of target traits. Three methods to improve the selection efficacy will be described in the following chapters.

2.3.1 Molecular Marker Assisted Selection

Molecular breeding tries to discover plant’s genes and their functions. The use of molecular markers allows plant breeders to screen large populations of plants and helps to select the genotypes possessing the trait of interest to be used to breed high-performing cultivars. The role of molecular markers in the characterization of medicinal plants was reviewed by Tharachand et al. (2012) and Sarwat et al. (2012). Several types of markers were used, including Randomly Amplified Polymorphic DNA (RAPD), Sequence Characterized Amplified Region (SCAR), Inter Simple Sequence Repeats’ (ISSRs), Amplified Fragment Length Polymor-phism (AFLPs), Single Nucleotide PolymorPolymor-phisms (SNPs), Simple Sequence Repeats (SSRs, or microsatellites), Restriction Fragment Length Polymorphisms (RFLPs).

In a recent study of Graham et al. (2010), the molecular basis for marker-assisted breeding ofArtemisia annua was established. Quantitative trait loci (QTL) related with key traits controlling artemisinin yield were identified. QTL analysis is a statistical method that links phenotypic data (specific traits) and genotypic data (molecular markers) to explain the genetic basis of complex traits. The presence of positive artemisinin yield QTL in parents will help to obtain new high-yielding hybrids for the future helping to answer more quickly to the increasing demand in artemisinin.

Molecular markers provide an independent and objective approach for the characterization of medicinal plant materials and can be used for an effective breeding. Furthermore, molecular markers can help to efficiently manage germ-plasm collections (in-situ/ex-situ) through monitoring and assessing genetic diver-sity, screening of germplasm collection for duplicates and formulating critical conservation strategies on what should be conserved on a priority basis. In the future it will be very fruitful if a concentrated effort was made to integrate existing molecular marker data and to co-ordinate projects of molecular characterization of medicinal plants.

2.3.2 Rapid Methods to Determine Valuable Compounds

One of the most important breeding aims is the improvement of the content of valuable compounds in the plants. However, the high expenses for phytochemical analysis are a limiting factor for breeding in medicinal and aromatic plants.

Therefore, rapid and cheap analytical methods are essential to provide a high selection efficiency by analysing an appropriate amount of individual plants. In addition, analytical methods are required allowing to analyse small sample quan-tities such as young single plants or parts of them without preceding sample

preparation. Furthermore, non-destructive methods are preferred to keep the inves-tigated plant material alive for following breeding procedures.

A rapid, low cost analytical method is near infrared spectroscopy (NIRS) (Schulz et al.1999; Camps et al.2011; Camps et al.2014). A NIRS-based method was developed to determine the artemisinin content in dry leaf powder of Artemisia annua (Camps et al.2011). In another study, hand-held NIRS (1,000–1,800 nm) measurements and FT-NIRS (1,000–2,500 nm) measurements were tested to deter-mine the essential oil content of oregano dried leaf powder. The FT-NIR approach allowed the development of an accurate model for the essential oil content predic-tion. Although the hand-held NIR approach is promising, it needs additional development before it can be used in practice. There are several other examples proving that near infrared and mid-infrared spectroscopy techniques are extremely helpful supporting very efficiently plant breeding programs and evaluation of plant resources (Schulz2010).

Another method for rapid analysis is the device SMart Nose®. The discriminat-ing power of the SMart Nose®, an electronic nose based on mass spectrometry of the gas phase, was tested on seven cultivars representing four chemotypes previ-ously distinguished by HPLC-DAD (Vouillamoz et al. 2009). This experiment shows that the distinction between the cultivars with the SMart Nose® is fast, reproducible and inexpensive. The use of the SMart Nose® could be extended in the future for screening of genotypes from natural populations and progenies after crossing to detect a particular chemotype.

On-site measurements on living plants can be performed with SPME (Solid Phase Microextraction) by placing a suitable glass chamber containing sidearms closed with septa over the plants. Depending on the ambient temperature approx-imately 15–20 min are needed for sampling. Then the loaded fibre can be directly analysed or mailed to a remote laboratory. SPME-GC field sampling of living rose flowers could be easily performed (Schulz2003) and allowed to discriminate the fragrance of different rose cultivars. This on-field non destructive method has been also successfully applied in breeding research to analyse numerous rose progenies of crossings with different smelling rose cultivars.

2.3.3 Rapid Determination of Resistance Against Biotic and Abiotic Factors

An analysis of 302 scientific publications on resistance research and breeding in medicinal and aromatic plants showed that the activities have been intensified in this field (Gabler2002). The majority, i.e. 70 %, of the contributions was concerned with fungal diseases, reflecting the economic importance of this pathogen group as a damaging factor. Rapid screening methods for resistance against fungal diseases are necessary to improve the breeding efficiency. A methodology tested by Michel et al. (2014) was focused on Anthracnose, a major production constraint for H. perforatum caused by the fungus Colletotrichum gloeosporioides (Penz.). A greenhouse screening method based on seedlings mortality was developed to

eliminate accessions susceptible to anthracnose in the early stage of breeding for resistant cultivars. The mortality of 22 accessions at the seedling stage artificially inoculated with a strain of C. gloeosporioides was positively correlated with the mortality in the field tests. Breeding of anthracnose resistance ofH. perforatum can be based on a greenhouse screening of seedlings to eliminate accessions susceptible to anthracnose in the early stage of breeding.

Resistance breeding in medicinal and aromatic plants is also focused on other biotic factors such as pests (nematodes and arthropods), as well as abiotic factors such as cold, heat, drought, wetness, nutrition deficiency or oversupply, saliniza-tion. Improvements in abiotic stress tolerance are very complex and can rarely be selected for in the greenhouses at seedling stage of the plants (Gabler2002).