In vitro soil-less (IVS) rooting medium

The principle hypothesis of this thesis is that hypoxia, in agar-based media, compromises rooting in vitro. From a practical point of view this is important because most plant tissue culture activities require the material to be successfully acclimatised in a nursery environment. Compromised rooting often results in excessive losses at this stage which are costly and inconvenient. In addition, many plants with commercial and/or scientific interest remain unavailable as they are not able to be rooted and acclimatised reliably. The use of agar as a rooting medium has limited the capacity of plant tissue culture to clonally propagate many plants. The thesis begins by demonstrating how poorly some plants respond to agar rooting media. Juvenile Chamelaucium hybrid microcuttings were pulsed with IBA 40 mcg M and then placed for 3 weeks on either M1 (1/2 MS) or aerated in vitro soil-less substrate (IVS) (Chapter 2). IVS had 42-82% rooting at the end of Stage 3 compared with 0-1% in agar. Shoot survival for IVS-rooted microcuttings was significantly greater than M1-rooted shoots. Pulsed shoots placed in IVS showed root primordia after 7 days. In contrast, shoots placed in agar showed no root primordia after 21 days and formed callus but did not root when subsequently placed in IVS for a further 4 weeks. The agar medium almost totally and permanently inhibited the capacity of competent shoots to form root primordia and roots. The effectiveness of different types of aerated and non-aerated media, including IVS, were tested to validate the hypothesis (Chapter 3). Microcuttings from shoot cultures of two Australian plants Grevillea thelemanniana and Verticordia plumosa x Chamelaucium uncinatum were pulsed for 7 days on a high auxin (40 mcg M IBA), agar-solidified medium in the dark. Rooting of the microcuttings was then compared on five experimental substrates: a) standard agar M1 medium (1/2 MS, no hormones, 8 g agar L-1), b) porous-agar medium (1/2 MS, no hormones, 30 g agar L-1, solidified then blended to provide aeration), c) white sand wet with liquid M1, d) white sand with M1 medium containing agar, and e) IVS. A separate experiment involved flushing the IVS soil profile with low or normal oxygen. Low and variable rooting percentages were recorded on the controls on M1 medium. Root induction and average total root length per microcutting at final harvest were significantly higher using the porous media including IVS, blended agar or white sand. The M1 medium and the addition of M1 medium to sand suppressed the percentage rooting and elongation. Flushing the IVS rooting medium with low oxygen also suppressed rooting. The experiments showed that increasing the air-filled porosity of the rooting medium has a positive effect on rooting and this is most likely due to the increased oxygen at the base of the microcutting. The role of ethylene, and the sugar and nutrients in M1 were not investigated. The efficacy of the IVS protocol on a range of Australian herbaceous and woody species was investigated to determine whether the observed benefits were generic or plant specific (Chapter 4). Improved rooting in IVS compared to agar was shown for 28 Australian species and genotypes from the families Liliaceae, Haemodoraceae, Myrtaceae, Thymelaeaceae, Proteaceae, Goodeniaceae and Rutaceae. Twenty-seven of the 28 species rooted in IVS medium at equal or better rates than in M1. In three cases - Actinodium cunninghamii, one of the Pimelea physodes genotypes and one of the Eriostemon australasius genotypes - shoots did not root in M1 but showed good root development in IVS medium. With few exceptions average root length and number in microcuttings rooted in IVS was superior to those in agar medium. To further test the resilience of the hypothesis, it was tested on nodal microcuttings of lentil which are recalcitrant to root in vitro (Chapter 5). The veracity of a published conclusion that inverted lentil microcuttings (with their base in the air) root better because of their altered polarity was also examined. It was found that, as is the case for many species, roots initiated and grew only at the proximal end of the microcutting regardless of its orientation. When the proximal end was in agar (a hypoxic environment) the rooting percentage was low (9-25%) even when the orientation of the microcutting was altered by inverting the culture tube. In contrast, when the proximal end of the microcutting was in an aerobic environment (from the shoot being placed upside down in agar medium or placed normally or upside down in an aerated medium) rooting percentages were higher (62-100%). Given that Stage 2 microcuttings are prepared with the objective to root and acclimatise them to nursery conditions, the duration of this activity becomes important as it can impact on plant quality and costs. The pulsing protocol and the length of time that Stage 3 cultures remain in the culture room during the rooting phase is a component of the unit cost of production of each rooted microcutting. Initially a 7-day IBA pulse was used after which the pulsed microcuttings were transferred to IVS to root. Chapter 6 shows that the pulsing period can be shortened to one day or replaced with a single auxin dip while still achieving high rooting percentages and maintaining plant quality. These materials handling improvements go some way to realising the logistical benefits of ex vitro rooting but without compromising the positive influences of hygiene and a stable environment of the in vitro environment.