Refining the application of direct embryogenesis in sugarcane: effect of the developmental phase of leaf disc explants and the timing of DNA transfer on transformation efficiency

A rapid in vitro protocol using direct somatic embryogenesis and microprojectile bombardment was investigated to establish the developmental phases most suitable for efficient sugarcane transformation. Immature leaf roll disc explants with and without pre-emergent inflorescence tissue were compared. It was shown that for effective transformation to occur, explants should be cultured for several days to allow initiation of embryo development prior to bombardment. Leaf roll discs with pre-emergent inflorescences showed a higher degree of embryogenic competence than non-flowering explants, and transformation efficiency was higher when explants containing floral initials were bombarded. Despite the occurrence of high numbers of phenotypically negative plants, combining the use of inflorescent leaf roll discs with direct embryogenic regeneration has the potential to improve the speed and efficiency of transgenesis in sugarcane.     

Elimination of virus and rapid propagation of disease-free sugarcane (<Emphasis Type="Italic">Saccharum</Emphasis> spp. cultivar NCo376) using apical meristem culture

The use of apical meristem culture for simultaneous virus elimination and shoot proliferation in sugarcane was assessed. Virus-free plants were propagated from Sugarcane mosaic virus and Sugarcane yellow leaf virus-infected material of the South African commercial cultivar, NCo376. A combination of thermotherapy by hot water treatment of stem sections (nodes) and subsequent germination of vegetative buds at 40°C and optimal meristem size were key factors for the production of virus-free plants. Only meristems of 2 mm in length or of a smaller size (but >0.5 mm) resulted in virus-free sugarcane. Shoot induction and proliferation via direct organogenesis were achieved on Murashige and Skoog nutrient medium supplemented with 0.1 mg l⁻¹ 6-benzyladenine and 0.015 mg l⁻¹ 6-furfurylaminopurine (KIN). The established protocol provides for the rapid proliferation of virus-free shoots from infected sugarcane plants and approximately 1,300 shoots were propagated from a single 2 mm meristem in 11 weeks. Plants remained virus-free when tested 12 months later.     

Assessment of Al3+ availability in callus culture media for screening tolerant genotypes of Cynodon dactylon

Aluminium-tolerant genotypes of Cynodon dactylon are potential candidates for the vegetation of gold mine tailings in South Africa. As a prerequisite to in vitro selection of tolerant genotypes, this work aimed at assessing and adapting micropropagation media to ensure Al³⁺ activity and toxicity. This was investigated using MINTEQA2, a chemical equilibrium speciation model. The maximum AlAl³⁺ activity achieved in any medium was 7.5 M. Of the seven published media investigated, four never achieved an activity greater than 4 M at 3–4 mM aluminium. The most appropriate medium was that of Yamamoto et al. (1996) (modified MS without KH₂PO₄ and EDTA), as it showed an increasing range of AlAl³⁺ activities from 2 to 7.5 M at aluminium concentrations from 0.25–2.5 mM. An improved modified MS formulation retaining phosphate was investigated because phosphate is an important component of our medium for callus induction in C. dactylon. Using MINTEQA2, no reduction in AlAl³⁺ activity by phosphate was detected in standard MS medium at pH 4. Through further simulations a new modified MS medium was derived with 1 mM SO ₄ ^2– and no EDTA at pH 4, which gave the maximum AlAl³⁺ activity (7.5 M) at 2 mM aluminium. This medium gave the highest AlAl³⁺ activities for the 0.25–2 mM concentration range of all the tested formulations, including the seven published media. It also resulted in significantly higher callus growth rates than standard MS media and other tested media. This new medium is currently being used to screen C. dactylon for aluminium tolerance at pH 4.     

Responses of meristematic callus cells of two Cynodon dactylon genotypes to aluminium

Responses to Al3+ of embryogenic callus cells of an Al-sensitive (Al-S) and Al-resistant (Al-R) Cynodon dactylon genotype were evaluated with regard to Al3+ toxicity and resistance. A chemical equilibrium speciation model (MINTEQA2) was used to ensure the availability of the Al3+ ion in culture media, which was supplied as 0.08-2.3 mM Al3+ for 2-8 weeks. Increasing Al3+ concentration and exposure time had a greater negative impact on the Al-S than on the Al-R genotype, in terms of callus growth rate and frequency of non-embryogenic cells. Exposure to 0.8 mM Al3+ for 2 weeks resulted in an 88% reduction in the Al-S meristematic cell number, whereas that of the Al-R genotype remained unaffected. In addition, the Al-S cells accumulated three times more Al in the nucleus than did the Al-R cells, suggesting that Al interfered with mitosis. The Al-R cells appeared to exclude Al3+ from its cells through an increase in extracellular pH (4.34 in Al-R and 4.08 in Al-S) and by the immobilisation of Al in the cell wall (33% more in Al-R). The results showed that by studying the cellular responses to Al3+ it is possible to discriminate between the Al-S and Al-R C. dactylon genotypes.     

An in vitro mutagenesis protocol for the production of sugarcane tolerant to the herbicide imazapyr

A protocol was developed to induce and identify imazapyr tolerance in sugarcane, which involved induction of somaclonal variation via exposure to 8 or 16?mM ethyl methanesulfonate for 4?h, followed by a stepwise increase in imazapyr concentration in the medium from 0.08 to 0.16???M. The regenerated plantlets were then acclimatized for 3?mo after which they were sprayed with 182?g?a.i.?ha^?1 imazapyr, and the above-ground biomass was determined after 47?d. Following a 1-mo waiting period for the putative tolerant plants to regrow, acetohydroxyacid synthase (AHAS; EC 2.2.1.6) enzyme assays of the plants that survived and showed a normal growth pattern were undertaken. Based on the enzymatic I ₅₀ values, three imazapyr-tolerant genotypes were identified with an AHAS activity of 2.8 to 4.0 times that in sensitive sugarcane plants.