Nemesia Root Hair Response to Paper Pulp Substrate for Micropropagation

1. Introduction

Micropropagation is a powerful biotechnology for plant multiplication [1, 2], but plant losses during acclimatization in greenhouse reduced, for some species, the asset of in vitro culture multiplication. In vitro rooting induction can be mediated by adding plant growth regulators or hormone-like substances to the culture medium [3]. However, the survival rate of these plants during acclimatization is low [3, 4]. In fact, greenhouse culture conditions like hygrometry, CO₂ levels, and nutrient bioavailability in culture medium are drastically different from those used for in vitro micropropagation. Most of the time, in vitro culture medium is composed of macro- and micronutrients, vitamins, carbohydrates, and eventually plant growth regulators gelified by polysaccharidic substances like agar. So, root formation in vitro could be drastically different from in classical greenhouse substrates. Gonçalves et al. [3] suggested that the lower survival rate during plant acclimatization is due to nonfunctionality of the in vitro developed rooting system. Root hairs constitute the major plant/substrate interface as they represent as much as 70% of the plant root surface [5, 6]. So, it could be assumed that root-hair nonfunctionality can drastically reduce water and mineral nutrient uptake, thus representing a limiting key step to acclimatization in peat substrate.

As first proposed by Afreen-Zobayed et al. [4] for sweet potato, paper pulp could be a potentially suitable substrate for in vitro culture and functional root-hair production. In order to clarify this assumption, this study compares in vitro development of an ornamental plant, Nemesia denticulata (Scrophulariaceae), on several substrates like agar and paper pulp. Moreover, enhancement of nemesia acclimatization through the use of paper-pulp substrate was evaluated.

2. Experimental Procedures

2.1. Preparation of Paper-Pulp Miniplugs

Paper pulp (a mixture of wood fibers from deciduous trees) was kindly provided by L. Harvengt from AFOCEL (http://www.fcba.fr/). Paper pulp was rehydrated in boiling water (200 g dry mass·L⁻¹) for 30 min and then vigorously mixed during 30 min in order to eliminate remaining aggregates. After supplemental water draining, paper pulp was manually pressed in plug molds ( $16\times 15$  mm, $Ø\times H$ ) and dried at 50°C for 24 h.

2.2. Plant Culture and Acclimatization

Nemesia denticulata (Scrophulariaceae) plants were cultivated on Murashige and Skoog’s (MS) modified by Van der Salm et al. [7] medium supplemented...