Adventitious shoot formation on leaf cuttings in vivo, a tool in horticulture

Adventitious shoot formation implies the regeneration or development of shoots from fully differentiated tissue. Its application has, after the rise of in vitro culture, assumed large proportions. Then the question arose whether in vivo adventitious shoot formation could not be applied more widely in commercial horticulture. To answer this question investigations were made on the regeneration of leaf cuttings and the results are presented in this thesis.It was found that the majority of a large number of plant species and cultivars was unable to regenerate shoots on leaf cuttings spontaneously. Various attempts to induce shoot regeneration with the cytokinin 6-benzylaminopurine (BA) were unsuccessful. BA had a positive effect, however, in a few species which regenerated shoots spontaneously (Chapter 1). These findings led to investigation of the effectiveness of plant growth regulator treatments to induce shoot regeneration in four species, Brassica oleracea , Lunaria annua , Nicotiana alata and Ruta graveolens , of which it was known that they were able to regenerate shoots, but which differed in the capacity to do so (Chapter 2). Cytokinins and auxins were applied prior to planting the cuttings as well as after roots had been formed. In all four species the treatments had marked effects. They fitted into two already known reaction patterns: that described by Skoog and Miller (1957) in which shoot regeneration is promoted at a high cytokinin/auxin ratio, and that described by Harris and Hart (1964) in which auxin is promotive for shoot regeneration. A new feature was that leaf cuttings from one and the same species showed both reaction patterns, as was found for Lunaria annua and Nicotiana alata . From these results it was concluded that regulator treatments still can be rather effective.As was shown in Chapter 1, simple applications of cytokinin were insufficient to induce shoot regeneration on leaf cuttings of. plant species with low regeneration capacity. Therefore the research was concentrated on one species, chrysanthemum ( Chrysanthemum morifolium ), of which an easy to regenerate genotype, cv. Bravo, and a difficult one, cv. Super Yellow, were used. Chapter 3 describes how shoot regeneration in 'Bravo' can be influenced by environmental factors. As a rule, at moderately high temperatures (13-17 °C) good results were obtained. Pretreatment with low temperature (9 °C) during the rooting phase had an additional favourable effect on the induction of shoot regeneration, whereas high temperature (21 - 25 °C) during the final phase accelerated the realization of the regeneration. Daylength of 8 h (SD) initially delayed the appearance of the adventitious shoots compared with daylength of 16 h (LD), but after some time the percentage of shoot formation was the same under both conditions. The leaf cuttings could not stand a high light intensity (>approx. 30 WM -2), which accelerated senescence and decreased shoot regeneration. Young leaves provided cuttings which regenerated better than those from old leaves, and leaves from vegetative plants were better than those from so-called crown bud plants, i.e. plants which have formed flower buds in LD and which, in general, show less vigorous growth.Shoot formation in 'Bravo' started about 18-20 weeks after planting, when the leaf cuttings began to senesce by degrees. This was called late shoot formation. The leaf cuttings from ' Super Yellow ' , studied under the same conditions as ' Bravo ' , never regenerated shoots. Cytokinin applied before planting gave shoot regeneration in both cultivars as early as nine weeks after planting; this was called early shoot formationApplication of auxin, notably indole-3-acetic-acid (IAA), together with cytokinin increased early shoot formation, yet the rate of success always remained very low under various experimental conditions. It was not possible either to establish the optimum concentrations of the regulators. However, low to moderately high temperature (9-17 °C) in the cutting benches was found to be an essential condition (Chapter 4).In a number of experiments it was shown that the developmental stage and pretreatments of the stock plants had a pronounced effect on early shoot formation of the chrysanthemum leaf cuttings (Chapter 5). Leaf cuttings from crown bud plants gave much higher percentages of early shoot formation than those from vegetative plants: around 85% at the optimum concentrations of the regulators (BA 6.25 - 12.5 mgl -1+ IAA 45 mgl -1). Small leaves from the inflorescences of plants which are about to start flowering also appeared to be very suitable. The most striking result was noted in leaf cuttings from plants which had stood for about a month in dim light (0.6-6 Wm -2). After 4-6 weeks a few dozens of adventitious buds per cutting arose, scattered over the entire length of the petiole. The pattern of this regeneration closely resembles that of regeneration in vitro. of these buds only a small part developed into plantlets. Moreover, many leaf cuttings were lost by rotting, particularly because they were very etiolated and tender as a result of the stock plant treatment.From the observation that the stock plant had such a strong influence a hypothesis was put forward regarding the process of shoot regeneration (Chapter 5 and General Discussion). In leaf cuttings from normally grown plants it is likely that regeneration inhibiting factors exist in the tissues surrounding the place of regeneration. Pretreatment of the stock plants reduces this inhibition and then the leaf cuttings can respond to shoot regeneration inducing factors like cytokinin, i.e. leaf cuttings are physiologically conditioned by the pretreatment. This hypothesis holds not only for early but also for late shoot formation where no regulators are applied just before planting the cuttings. In late shoot formation the physiological conditioning only starts when the leaf cuttings are gradually senescing. Moreover, this hypothesis explains why in vitro regeneration generally is easier than the in vivo regeneration described here: in the small explants commonly used in vitro there is only a low amount of surrounding tissue left which can inhibit shoot regeneration. Thus the small size of the in vitro explants will have the same effect as the stock plant pretreatment in the case of in vivo regeneration. From a practical point of view, however, excision of small explants is much easier than pretreatment of whole plants. Therefore, it was concluded that in plant species with a low to moderately high regeneration capacity, like in chrysanthemum studied here, in vivo shoot regeneration cannot match the results in vitro.For practical application, in vitro shoot regeneration should be preferred to in vivo regeneration in the case of recalcitrant species. For species with a high regeneration capacity, however, it is not so clear which of the two procedures has to be chosen. Shoot regeneration in plants with such a high regeneration capacity is described in Chapters 6 and 7; it concerns certain 'Elatior' -begonia (Begonia x hiemalis ) cultivars. In general with Begonia spp. SD and low temperature promote shoot regeneration, but literature on 'Elatior'- begonias contains different views on this aspect. Our experiments showed that only a short period of SD (4-14 days), applied either to the stock plants or to the cuttings, was favourable, promoting the early phase of adventitious bud initiation. A long period of SD (>28 days) applied to the cuttings had an adverse effect on the growth of these buds; no shoots emerged from the soil. Low temperature had not the same effect as SD and shoot regeneration was generally better when high temperatures (21-25 °C) were used in the cutting benches (Chapter 6).The effects of regulators on regeneration in Begonia spp. generally fit well into the reaction pattern as described by Skoog and Miller (1957): cytokinin promotes shoot regeneration and auxin root regeneration. When applied to the 'Elatior'- begonias, however, BA had an adverse effect; the number of initiated adventitious buds was increased, but shoot growth and development were completely disturbed and inhibited. A low auxin concentration (IAA 0.2-1 mgl -1) promoted adventious shoot formation. Combination of high BA concentration (25 mgl -1) with high IAA concentration (5-25 mgl -1) led to regeneration of many buds, distributed all along the petiole, but only a small number developed into shoots (Chapter 7).It was supposed that with the 'Elatior'-begonias the investigated factors influence mainly the last phases of the regeneration process, bud initiation and shoot development. The earlier phase of shoot regeneration induction proceeds rapidly and presents no problems. So in this respect there is no reason to change over to in vitro procedures.Finally, from the results presented in this thesis conclusions were drawn concerning efforts to attain a wider application of in vivo adventitious shoot formation in horticultural practice. On the one hand such efforts are very meaningful for species with a high regeneration capacity, on the other hand they are not for species with a low capacity. For the latter, in vitro procedures can be used with a much better result than the in vivo procedures