D. Blakesley

Auxin Metabolism and Adventitious Root Initiation

In this chapter I will report on and discuss the role of auxins in adventitious root initiation, particularly the relations between endogenous indole-3-acetic acid (IAA) and the early events of adventitious rooting. The fact that IAA is involved is well established, although much of the data to support this is circumstantial. It will be not possible in this review paper to describe all the work on auxin application, transport and metabolism that might be relevant to studies of adventitious root initiation. Evidence derived from studies on auxin application has been reviewed many times [e.g., Audus (1959) and Blakesley et al. (1991b)] and will not be considered in detail in this paper. Evidence from more recent work on the analysis of endogenous auxin, and from studies on transgenic plant tissue, will be germane to the present paper. From these studies it will be apparent that we still do not have a clear understanding of the exact role of auxin in the process of adventitious root initiation. The aim of the latter part of this paper will be to describe briefly the newer technologies which are available to plant developmental physiologists, and to indicate new directions for researchers to approach the problem of auxin involvement in adventitious root initiation.

Uptake and metabolism of 6-benzyladenine in shoot cultures of a range of species

Shoots of a range of species were cultured in vitro on a medium containing 2.22 μM benzyladenine (BA). After 25 days in culture the amount of BA taken up differed between species. The conjugates of BA present in the plant tissues were identified by HPLC. The pattern of BA metabolism varied considerably between species as did the actual levels of each conjugate. Benzyladenine accumulated in Hippeastrum, Alstroemeria and Ficus with little conjugation. In Musa, Rhododendron and Fuchsia BA also accumulated along with its ribosyl and glucoside conjugates. In contrast, very low levels of BA were detected in Gerbera and Dendranthema, but various glucosides did accumulate.

Uptake and metabolism of 6-benzyladenine in shoot cultures of Musa and Rhododendron

Shoots of Musa and Rhododendron were cultured in vitro on a medium containing 0.5 mg l-1 BA. Shoots growing in the presence of [14C]BA were harvested at intervals during the culture period. Uptake of BA was linear throughout this culture period in Musa but slowed considerably in Rhododendron shoots after day 10. Rhododendron shoots absorbed 40% of the BA present in the medium and Musa shoots absorbed 52%. In each species the principal metabolite formed was [9G]BA. Benzyladenine was present in significant levels only in the pseudostem of Musa.

Optimisation of somatic embryogenesis in fourteen cultivars of sweet potato [Ipomoea batatas (L.) Lam.]

Abstract Culture procedures have been developed to facilitate the induction and maintenance of somatic embryogenic tissues in 14 out of 16 tested cultivars of sweet potato [Ipomoea batatas (L.) Lam]. Both the size of the axillary bud explant and the type of auxin were found to be critical for the successful induction of somatic embryogenesis. Of the five auxins screened 2,4-dichlorophenoxyacetic acid 2,4-D and 2,4,5-trichlorophenoxyacetic acid were the most effective, with use of the latter inducing the production of embryogenic tissues in 7 cultivars which responded poorly or not at all to 2,4-D. Procedures for secondary/cyclic embryogenesis, formation of mature embryos and their conversion to plants are also described.

Cryopreservation of non-encapsulated embryogenic tissue of sweet potato (Ipomoea batatas).

Embryogenic tissue of the sweet potato (Ipomoea batatas (L) LAM) genotype TIB 10 was established from in vitro axillary shoot tips on Murashige and Skoog (1962) medium supplemented with 5 μM 2,4-dichlorophenoxyacetic acid. Embryogenic aggregates of fresh mass 9.0–12 mg were subjected to a rapid freezing protocol in liquid nitrogen following sucrose preculture and varying degrees of dehydration. Up to 50% of embryogenic explants survived rapid freezing after preculture on 0.4 or 0.7M sucrose only. Dehydration with silica gel to moisture contents in the range 18–41% improved the survival after cryopreservation of embryogenic tissue. Tissue dehydrated for intermediate periods exhibited poor survival. Following freezing, embryogenic tissue appeared to develop normally, retaining its competence to produce mature embryos and plantlets.

Cryopreservation of embryogenic tissue of a range of genotypes of sweet potato (Ipomoea batatas [L] Lam.) using an encapsulation protocol

Abstract Embryogenic tissue of nine sweet potato [Ipomoea batatas (L.) Lam] genotypes from Asia, Africa and the Americas was established from in vitro axillary buds on Murashige and Skoog medium supplemented with 2,4-dichlorophenoxyacetic acid or 2,4,5-trichlorophenoxyacetic acid. Embryogenic aggregates, 1.0–2.0 mm in diameter, were encapsulated in alginate gel, precultured on medium containing elevated levels of sucrose and dehydrated prior to rapid freezing in liquid nitrogen. The maximum survival of embryogenic tissue ranged from 4% to 38%, depending on the genotype. With the incorporation of a slow-cooling step, survival was generally much higher than that obtained after rapid freezing alone. Five of eight genotypes tested with this protocol gave survival percentages in excess of 55%, and a further two in excess of 33%, all after evaporative dehydration. The most effective sucrose treatment(s), however, varied with the genotype.

Cryopreservation of embryogenic tissue of sweet potato (Ipomoea batatas): use of sucrose and dehydration for cryoprotection

Embryogenic tissue of two sweet potato (Ipomoea batatas (L) LAM) genotypes, TIB 10 and Nemanete (Nem), was established from in vitro axillary meristems on Murashige and Skoog (1962) media supplemented with 2,4-dichlorophenoxyacetic acid and 2,4,5-trichlorophenoxyacetic acid respectively. Embryogenic aggregates of approximately 1.5–2.0 mm in diameter were subjected to a rapid or a two-step freezing protocol in liquid nitrogen following alginate encapsulation, sucrose preculture and varying degrees of dehydration. Up to 28% of encapsulated embryogenic aggregates of TIB 10 survived rapid freezing without dehydration. This was not enhanced by dehydration prior to freezing. However, survival after dehydration was enhanced up to 74% by incorporating an initial slow cooling step prior to plunging the tissue into liquid nitrogen. Following freezing, embryogenic tissue appeared to develop normally and retained its competence to produce mature embryos and plantlets. Similar results were obtained with Nem, although the survival percentages were much lower.

Shoot regeneration from mature endosperm of Passiflora foetida

Murashige and Skoog (1962) medium supplemented with 2 μM 6-benzylaminopurine (BA) induced adventitious shoots on mature endosperm explants, whilst gibberellic acid (GA3) and casein hydrolysate stimulated growth and development of these shoot primordia. Plantlets were successfully weaned in vivo. These plants were found to be triploid and flowered, although fruit set was not observed.

The regeneration and screening of watercress somaclones for resistance to Spongospora subterranea f.sp. nasturtii and measurement of somaclonal variation.

A range of watercress (Rorippa nasturtium-aquaticum) explants (stems, hypocotyls, true-leaves, cotyledons and petioles) were tested for their capacity to regenerate adventitious shoots from callus formed using Murashige and Skoog medium containing different concentrations of thidiazuron and 2,4-dichlorophenoxyacetic acid. The highest shoot regeneration rate was a mean of 18 shoots per responding explant from stem callus formed on medium containing 5 μM thidiazuron and 0.05 μM 2,4-dichlorophenoxyacetic acid. A histological study confirmed that shoots originated directly from callus tissue. Twenty five percent of somaclones exhibited somaclonal variation in leaf shape, plant height, axillary branching or ploidy. The variation in 6% of somaclones was heritable to the first selfed generation. A screening protocol was developed to permit the identification of somaclones with increased resistance to the economically damaging watercress root pathogen, Spongospora subterranea f. sp. nasturtii. Although 883 somaclones were screened using this protocol, no significant increase in disease resistance was detected.

Benzyladenine ribosylglucoside: A metabolite of benzyladenine in Gerbera jamesonii

Abstract 6-benzylamino-9-(glucosylribosyl)purine was identified as the major metabolite of 6-benzylaminopurine in petioles plus apices and callus of 24-day-old shoot cultures of Gerbera jamesonii .