M. A. Norton

Micropropagation of guava (Psidium guajava L.)

Summary Guava (Psidium guajava L.) is difficult to propagate using conventional asexual techniques, with most growers using seedling planting stock. However, these seedlings are highly variable. We therefore developed an in vitro technique to clonally propagate guava. Various concentrations of BAP (6-benzylaminopurine) and TDZ (thidiazuron) were used to regenerate and micropropagate plants. Two explant sources were compared: greenhouse grown plants (GHRP) and in vitro-harvested axillary buds (IVDS). GHRP with BAP at 2 mg l–1 gave 3.7 shoots per single node cutting with an average length of 0.7 cm. Shoots 3.0 cm long were obtained with 0.5 mg l–1 BAP, however only 2.1 shoots per explant were produced. For IVDS, the largest number of shoots (3.9 per explant) was obtained with BAP at 0.25 mg l–1 , with an average shoot length of 1.6 cm. Generally, lower concentrations of BAP gave fewer but longer shoots. The highest number of roots and longest roots per shoot (5.4 and 2.0 cm, respectively) were obtained with 1 mg l–1 indole-3-butyric acid (IBA). A protocol for producing clonal plants over eight weeks is described.

The Use of Thermotherapy and in vitro Meristem Culture to Produce Virus-Free ‘Chancellor’ Grapevines

ABSTRACT Grapevines (Vitisspp.) are very susceptible to virus diseases. Virus infection reduces fruit yield and quality. The objective of this work was to determine the usefulness of thermotherapy (37.2°C) and in vitromeristem culture to obtain virus-free grapevine plants cv. ‘Chancellor’. Grapevine leafroll-associated virus 1,3(GLRaV-1, 3) infected grapevines were multiplied in vitrofrom two infected mother-plants in half strength Murashige and Skoog medium (1/2MS) supplemented with 0.5 mg/L of BA and the in vitroplants were initially tested by ELISA to confirm their virus status; subsequently, 96 infected in vitroplants were propagated on 1/2MS medium with BA and subjected to 0, 7, 10, 12, 14, 16, 18, 20 or 22 days of exposure at 37.2°C. Afterward, the apical meristems from the plants surviving the thermotherapy treatment were excised and transferred to fresh 1/2MS medium with 0.5 mg/L of BA and grown in a culture room until they developed into entire plants. Control plants and all the plants that survived thermotherapy were assessed for their virus status using both ELISA and RT-PCR. After 20 days of exposure at 37.2°C, 100% of the plants submitted to thermotherapy were found to be virus-free by RT-PCR and ELISA tests. Plants derived from meristems with two or three primordial leaves remained virus infected. However, when meristem culture was combined with thermotherapy (12 or more days of heat treatment), all the meristem-derived plants were virus-free.

Establishment of contaminant-free perennial plants in vitro

SummaryPerennial plant tissue cultures are established by disinfecting field or greenhouse-grown plant parts and transferring them to sterile medium in vitro. Typically, shoots harvested from field or greenhouse-grown plants are placed in water, either to force growth from dormant branches or to maintain them until ready for explanting. In spite of extreme care, 90 to 100% contamination rates in newly established in vitro cultures are not unusual. Experience has identified several routine procedures that reduce contamination, such as minimizing the amount of time a stem cutting is maintained in water before being explanted, adjusting pH of the medium to a more acidic condition, and using pH neutralized bleach to sterilize instruments during subculture. Other methods to reduce contamination include establishing field-grown plants in a greenhouse where inoculum levels can be better controlled, trellising vining plants to get them off the soil, avoiding wetting foliage, and selecting vigorous explants that are not in contact with soil.

Leaf morphology affects horseradish regeneration in vitro.

The morphology of horseradish (Armoracia rusticana P. Gaertn., B. Mey & Scherb.) leaves varies through the growing season. The leaves range from laminate (complete) in the summer to pinnate (fern-leaf) toward the end of the growing season in the fall, with intermediate types appearing regularly. The causes of these changes are not understood. To determine whether leaf morphology affects their ability to regenerate adventitious shoots in vitro, laminate, pinnate, and intermediate leaves of 10 horseradish cultivars were established in vitro. Laminate leaves were more likely to regenerate shoots (77%) than other leaves (23%). It is recommended that laminate leaves be used as explants to regenerate horseradish plants in vitro.

Regeneration and proliferation of pathogen-free horseradish (Armoracia rusticana) plants in vitro.

Summary Horseradish (Armoracia rusticana Gaertn.) leaf explants were tested for their ability to regenerate and proliferate shoots in vitro. Leaf explants were grown on Murashige and Skoog (MS) medium supplemented with different concentrations of either naphthalene acetic acid (NAA) or benzyladenine (BA), or a combination of both. The optimum number of regenerated shoots occurred at 15 µM NAA, while the optimum number of proliferated shoots occurred at 2 µM BA. Higher concentrations of BA resulted in reduced shoot height, inhibited rooting, and increased leaf deformation. A sample (1,000 plants) of both regenerated and proliferated plants was rooted, acclimatised to greenhouse conditions, then field-planted in two subsequent years. All plants survived. The ability of ten virus-free (VF) and ten virus-infected (VI) horseradish cultivars to regenerate shoots in vitro was tested. Nine of the ten VF cultivars regenerated more vigorous and healthier shoots than VI plants. The results of these experiments represent the first steps towards establishing a certified root programme to provide horseradish growers with large numbers of pathogen-free plants to restore yields of good quality roots.