Chapter 3: Micropropagation of three Brachystelma species
3.3 Results and discussion
3.3.4 Ex vitro rooting and acclimatization of B. ngomense, B. pulchellum and B
green house survival compared to shoots previously potted directly after in vitro root induction. During acclimatization, the shoots derived from this experiment had an extended survival period of beyond 10 weeks as opposed to the 7 week survival of the in vitro root induction treatments. This extension can be attributed to the ex vitro root induction that was observed by the fourth week under green house conditions. The survival rate after 4 weeks was 42%, 35% and 30% for B. ngomense, B. pulchellum and B. pygmaeum, respectively. By the end of the tenth week, survival rate was 5%, 0% and 3% for B. ngomense, B. pulchellum and B. pygmaeum, respectively. The decrease in survival, in this current study, is primarily attributed to the poor rooting which is commonly observed in micropropagation systems (DEBERGH AND READ, 1991; XIAO et al., 2011). The few surviving plants were observed to maintain a healthy green appearance with no obvious morphological abnormalities for both the above and below ground organs (Figure 3.10 C&F). The healthy green appearance was perceived as an indication of functional photosynthetic capacity. Subsequently, B. pygmaeum in vitro derived shoots were used to test the effect of pulse treatment with 100 mg/L of IBA at different time intervals (3, 12 and 21 min). By the end of 8 weeks, survival rate was 5% for 3, 12 and 21 min while there was no survival (0%) for the control. Thus, the exposure time had no effect on ex vitro root induction and overall survival of shoots remained low. Some studies
48 have shown that increasing exposure time promotes rooting (MADHULATHA et al., 2004). In a study by PHULWARIA et al. (2013), pulse treatment with IBA (100 mg/L) for 3 min was particularly effective in ex vitro root induction for Ceropegia bulbosa. Thereafter, 100%
rooting was observed which was followed by successful hardening and transfer to the field (PHULWARIA et al., 2013). In some instances, the same treatment will not reproduce the same result and there are many possible reasons for this which are not only limited to the differences of plant species. For example, MAKUNGA et al. (2005) found that the exact same IBA treatment used on the same plant (Thapsia garganica), possibly derived from one stock plant, reproduced a completely different result. Even though the pulse treatment used in this study did not yield a positive result, ex vitro rooting is known to be more advantageous in comparison to in vitro rooting especially for plants that are difficult to root (DEBERGH AND
READ, 1991; DOBRÁNSZKI AND TEIXEIRA DA SILVA, 2010; PHULWARIA et al., 2013).
The sequence of root and tuber induction is variable in the in vitro and in vivo conditions. In all the in vitro culture experiments, miniature tubers (Figure 3.2A) formed on cultured shoots often preceding root induction regardless of media composition whereas tubers and roots occur simultaneously in vivo. It is not clear at present whether the induction of in vitro tubers in the absence of a root system might have hindered root induction. Therefore, this “tuber before root” in vitro observation could well be a growth/morphological abnormality.
Morphological abnormalities are a common occurrence in micropropagated plants (SMULDERS AND DE KLERK, 2011; VANDEMOORTELE, 1999; WU et al., 2009; ZHAO et al., 2005b). In vitro abnormalities observed in this study include hyperhydricity (Figure 3.2C) and “bushiness” (Figure 3.10J) in the form of an abundance of small leaves on a short node section (D’ARTH et al., 2002; WHITEHOUSE et al., 2002). Hyperhydricity mainly affected adventitious shoots derived from callus. Shoots derived from nodal explants were seldom affected by hyperhydricity. No further morphological differences were observed between the parent plants and regenerants.
Concerning acclimatization incompetence, there are a couple of factors that have been reported as possible contributors. Light stress is one of the possible contributing reasons for this short-lived survival as transfer of plantlets to in vivo conditions of higher light intensities is known to have an overwhelming effect (CHANDRA et al., 2010; DEBERGH AND READ, 1991). Humidity is another factor that changes drastically, thus affecting plantlet survival (CHANDRA et al., 2010). Successful establishment of plantlets ex vitro, as a concluding step,
49 is crucial in micropropagation because many biotechnological applications are dependent on plant regeneration efficiency (TITOV et al., 2006; XU et al., 2009; YANG AND YEH, 2008;
ZHANG et al., 2001). Poor survival during acclimatization is often attributed to the heterotrophic mode of nutrition under which the plantlets develop morpho-physiological disorders such as poor control of water loss (DEBERGH AND READ, 1991; XIAO et al., 2011;
YANG AND YEH, 2008). However, Brachystelma has been observed to be a naturally fragile group of plants which might very well be a factor contributing to their poor performance in root development and acclimatization. Perhaps a study involving seed germination may yield better results and also shed some light on the rooting process of Brachystelma species.
During this study, it was quite challenging to obtain Brachystelma seeds. A few seed companies and nurseries such as Blackwood’s (Pietermaritzburg), Lifestyle seeds (Bloemfontein), Seedroom (SANBI) (Kirstenbosch), and Silverhill (Cape Town) were consulted but they had no supply of any Brachystelma seeds.
50 Figure 3.10: Micropropagation process of A-B. ngomense, D-B. pulchellum and G-B.
pygmaeum. A, D & G-Stock plants. B, E & H- In vitro establishment of B. ngomense, B.
pulchellum and B. pygmaeum .C- Fully acclimatized B. ngomense. F- Fully acclimatized B.
pygmaeum. I- In vitro rooting. J- Morphological abnormality of in vitro grown Brachystelma.
L- Brachystelma species in potting trays in mist house. M- Brachystelma species growing in green house. N- Humidity cover retaining moisture during first week of acclimatization in green house. O- Ex vitro derived roots. Scale bar = 10 mm.