Priya A. Joyce

Culture-independent survey techniques are fundamental tools when assessing plant microbiomes. These methods rely on DNA that is carefully preserved after collecting samples to achieve meaningful results. Immediately freezing samples to -80°C after collection is considered one of the most robust methods for preserving samples before DNA extraction but is often impractical. Preservation solutions can solve this problem, but commercially available products are expensive, and there is limited data comparing their efficacy with other preservation methods. In this study, we compared the impact of three methods of sample preservation on plant microbiome surveys: 1) RNAlater^®, a proprietary preservative; 2) a home-made salt-saturated dimethyl sulfoxide preservation solution (DESS); and 3) freezing at -80°C. DESS-preserved samples, stored at room temperature for up to four weeks, did not show any significant differences to samples frozen at -80°C, while RNAlater^® inflated bacterial alpha diversity. Preservation treatments did not distinctively influence fungal alpha diversity. Our results demonstrate that DESS is a versatile and inexpensive preservative of DNA in plant material for diversity analyses of fungi and bacteria.

Biolistic transformation is one of two popular methods for introducing genes into sugarcane. However, unlike Agrobacterium-mediated transformation, the efficiency of gene transfer into sugarcane cells, using the biolistic method is very high. In addition to this, the biolistic transformation method is independent of the explant genotype or tissue. It also has the advantage that a minimum DNA sequence of linearized plasmid can be used, thus eliminating the introduction of undesirable plasmid derived genes, delivering low-copy transgenic events. In this chapter, we describe the method for efficient delivery of genes into sugarcane cells using a biolistic approach.

Summary: Future genetic improvement of sugarcane depends, in part, on the ability to produce high‐yielding transgenic cultivars with improved traits such as herbicide and insect resistance. Here, transgenic sugarcane plants generated by different transformation methods were assessed for field performance over 3 years. Agrobacterium‐mediated (Agro) transgenic events (35) were produced using four different Agrobacterium tumefaciens strains, while biolistic (Biol) transgenic events (48) were produced using either minimal linearized DNA (LDNA) transgene cassettes with 5′, 3′ or blunt ends or whole circular plasmid (PDNA) vectors containing the same transgenes. A combined analysis showed a reduction in growth and cane yield in Biol, Agro as well as untransformed tissue culture (TC) events, compared with the parent clone (PC) Q117 (no transformation or tissue culture) in the plant, first ratoon and second ratoon crops. However, when individual events were analysed separately, yields of some transgenic events from both Agro and Biol were comparable to PC, suggesting that either transformation method can produce commercially suitable clones. Interestingly, a greater percentage of Biol transformants were similar to PC for growth and yield than Agro clones. Crop ratoonability and sugar yield components (Brix%, Pol%, and commercial cane sugar (CCS)) were unaffected by transformation or tissue culture. Transgene expression remained stable over different crop cycles and increased with plant maturity. Transgene copy number did not influence transgene expression, and both transformation methods produced low transgene copy number events. No consistent pattern of genetic changes was detected in the test population using three DNA fingerprinting techniques.