The components of plant tissue culture media II. Organic additions, osmotic and pH effects, and support systems

Management of plant virus and viroid diseases is a matter of vital importance and concern to the farmer, horticulturist, forester and gardener. It is well established that the virus and viroid diseases in different crops cause enormous losses in terms of quantity and quality of products (see Chapter-3 of Volume-I) (Sastry 2013b). Because of the serious losses they cause to agriculture and horticulture, virus/viroid diseases have acquired great importance in the realm of plant pathology and call for effective management measures against them. These diseases are not amenable to control by direct methods. Unlike fungicides and bactericides, no commercial viricides have yet been developed. However a number of indirect control measures for different crop plant-virus/viroid pathosystems suitable to varied agro ecosystems have been developed with an aim to avoid the pathogen or to minimize their impact on crop yields (Thresh 2003, 2006). As these diseases routinely cause systemic infections and have very effective methods of transmission, preventing their spread has become a complicated problem. Some diseases like Tobacco mosaic virus (TMV), Potato virus X (PVX), Cucumber green mottle virus (CGMMV), Citrus exocortis viroid (CEVd) and Potato spindle tuber viroid (PSTVd) are highly contagious and affect large number of hosts belonging to several families.

Management of Virus and Viroid Diseases of Crops in the Tropics

Friable embryogenic callus and somatic embryos of 4 Gladiolus cultivars were obtained on Murashige and Skoog (MS) medium with various concentration of auxins from the following explants: corm slices, young leaf bases and whole, intact plantlets. Somatic embryos transferred on MS hormone-free medium regenerated into plantlets. All plantlets obtained through embryogenesis did not differ phenotypically from the parental clones. The embryogenic friable callus has been maintained for over 2 years in culture and has retained a very high regeneration capacity.

Somatic embryogenesis and plant regeneration of Gladiolus (Gladiolus hort.).

Plant biotechnology in South Africa: Micropropagation research endeavours, prospects and challenges

Three cultivars (cvs.) of Gladiolus hybridus Hort., namely ‘Her Majesty’, ‘Aldebaran’ and ‘Bright Eye’ were successfully micropropagated. The cultures were established using intact cormels or segments of cormels and inflorescence axes on Murashige and Skoog (1962; MS) medium. The response depended on media supplements; both callus formation or direct induction of shoot buds was observed. Shoot differentiation from callus could be obtained on MS medium containing 1.0 μM BA (6–benzyladenine) and 10.0 μM NAA (α-naphthalene acetic acid) in all three cultivars. The same could be achieved by giving a heat shock (HS; 50 °C, 1h) to callus cultures (in case of ‘Her Majesty’ and ‘Aldebaran’ only) maintained on the basal medium. In these two cultivars, high sucrose concentration (0.232, 0.290 or 0.348 M) also favoured growth and proliferation of shoot cultures on a plant growth regulator-free medium at 20 °C in comparison to the cultures kept at 25 °C. On the other hand, shoot cultures maintained on the basal medium at 25 °C containing normal (0.058 M, i.e., 2.0%, w/v) sucrose concentration responded similar to those maintained at 20 °C on a high sucrose medium; reduced response was observed on normal sucrose containing medium at 20 °C. Heat shock enhanced shoot proliferation in the cultures maintained on basal medium, but induced prolific rooting in shoot cultures, within 5 days of HS, on high sucrose (optimum 0.232 M) medium. While the number of roots increased at higher sucrose concentrations in the medium in case of cvs. ‘Her Majesty’ and ‘Aldebaran’, the same was found to be independent of sucrose concentration in cv. ‘Bright Eye’. Generally the rooted plants produced on high sucrose (0.232 M) medium in comparison to medium with normal sucrose concentration showed better survival (ca. 90% as against 40%) in the soil.

In vitro propagation of Gladiolus hybridus Hort.: Synergistic effect of heat shock and sucrose on morphogenesis

Tissue culture techniques were applied to study the regeneration and growth of bulblets from bulb scale segments ofCrinum macowanii Bak. (bush- or march lily)in vitro. Shoots were induced on twin scales taken from the basal plate region of flowering-size bulbs on Murashige and Skoog (MS)-medium containing 0–20 mg l−1 NAA and BA and a modified MS medium (MMS medium) containing 1.25 mg l−1 ancymidol (A-RestTM), 0.1 mg l−1 NAA and 0.1 mg l−1 kinetin (ANK). Large bulblets could only be initiated on the latter. Subsequently the bulblets of 5 mm or more in diameter were trimmed and split in half, and secondary plantlets were regenerated on MMS-medium containing ANK or MS-medium without any growth regulators which in turn grew into bulblets suitable for splitting within 12–16 weeks. A total of 700–1000 bulblets could be obtained from each initial bulb within 12 months. Anatomical studies showed that the shoots were initiated from the epidermis and hypodermis on the abaxial surface of the meristematic tissue of the basal plate of the bulb scale. This technique is useful for the multiplication and preservation of a genotype, since plantlets regenerated in this manner should be genetically uniform.

Regeneration of bulblets from twin scales ofCrinum macowanii in vitro

The ability of the summer flowering Gladiolus dalenii Van Geel and the winter flowering G. tristis L. to form corms in vitro was investigated. G. dalenii spontaneously formed corms on a shoot induction medium consisting of the basal medium of Murashige & Skoog (1962) with up to 2.0 mg l-1 benzyladenine (BA), 3% sucrose and solidified with 2 g l-1 Gelrite®. The effect of different BA and sucrose concentrations as well as different temperatures on in vitro corm production of G. tristis was further investigated. The best production of shoots per explant was achieved on a medium containing 0.5 to 1.0 mg l-1 BA, sucrose concentrations of 6 to 9% and cultured at 15°C. The best corm production was achieved at the same temperature and with the same medium with the exception that BA was omitted from the medium. To test the effect of the osmotic potential on the formation of shoots and corms, sucrose was substituted by mannitol at various concentrations. Sucrose proved to be essential for both shoot and corm production and the use of mannitol had no beneficial effect.

In vitro corm production of Gladiolus dalenii and G. tristis

Amaryllis belladonna L. plants were multiplied successfully by means of tissue culture techniques. Different plant parts were tested as explant material, but plantlets could only be generated from the twin-scales and immature scapes. These in vitro-formed plantlets were divided into four parts and used for further multiplication. The twin-scale explants had the highest multiplication rate when a medium with 22.2 μM benzyladenine and 0.54 μM naphthaleneacetic acid was used. The sucrose concentration played an important role in the initiation of new plantlets, and the best results were obtained when a sucrose concentration of 2–3% was used. Anatomical observations were made during the initiation of the new plantlets.

In vitro propagation of Amaryllis belladonna

Adventitious shoots and plantlets were regenerated in vitro from floral stem explants of Crinum macowanii (Bak.) (bush lily). The length (age) of the floral stem as well as the orientation and position of the explant disc in the floral stem were the most important factors affecting shoot regeneration. The highest number of shoots were regenerated when immature floral stems of 70–100 mm were used as starting material, using the middle or basal parts of the stem, and orientating the discs with their proximal ends on the medium. Combinations of kinetin (4.65 μM) and either indoleacetic acid (0.57 μM) or naphthaleneacetic acid (0.54 μM), or a combination of benzyladenine (4.44 μM) and 2,4-dichlorophenoxyacetic acid (0.45 μM) resulted in the highest numbers of shoots being regenerated. Although a slight degree of callus formation was noticed on the cut-edges of the discs, shoot formation did not occur via callus, but directly from the floral stem epidermis. Unrooted shoots were rooted on MS-medium containing 0.17 M sucrose.

Adventitious in vitro plantlet formation from immature floral stems of Crinum macowanii

Virus-indexed Ornithogalum ev. Rojel plants were produced by eliminating Ornithogalum mosaic virus (OMV) through meristem-tip culture. The best plantlet regeneration was obtained from meristems derived from adventitious buds which developed on leaf explants taken from mother plants at the flowering stage. Acyclovir had no effect as an anti-viral compound on plantlet regeneration or virus elimination. Adenine arabinoside retarded plantlet development at concentrations of 10 mg l-1 and higher, while 5.0 mg l-1 suppressed the virus concentration beneath a detectable level in young plants. All the mature plants, however, tested positively for the presence of OMV.

https://link.springer.com/content/pdf/10.1007/BF00036146.pdf

D. I. Ferreira

Papers

Regeneration of bulblets from twin scales ofCrinum macowanii in vitro

In vitro corm production of Gladiolus dalenii and G. tristis

In vitro propagation of Amaryllis belladonna

Adventitious in vitro plantlet formation from immature floral stems of Crinum macowanii

Elimination of ornithogalum mosaic virus in the Ornithogalum cv. Rogel through meristem tip culture and chemotherapy