Maturation refers to the age-related progression in phenotypic expression of characters during plant development. Several models have been discussed (Greenwood 1992). The changes are epigenetic, are totally reversed during sexual reproduction, and maturation of apices is therefore in principle reversible (Timmis & Ritchie 1988), although clearly more stable in woody species than in many other plants (Hackett 1992).
There is a substantial literature, extending back over 25 years, reporting in vitro manipulation of the maturation state, in particular “rejuvenation”. The development of juvenile morphological characters on mature explants cultured in vitro has been observed in a number of horticultural species, including azalea, blueberry, apple, grape, mulberry, elderberry and raspberry (Brand & Lineberger 1992a). Forest tree species for which the expression, in cultured adult explants, of more juvenile states with respect to features such as morphology and rooting (most commonly), vigour, multiplication rate and orthotropism has been reported include Sequoia sempervirens (Fouret et al. 1984, Franclet et al. 1987, Franclet & Franclet-Mirvaux 1992), Sequoiadendron giganteum (Monteuuis & Bon 1989), Pseudotsuga menziesii (Timmis & Ritchie 1988), Betula papyrifera (Struve & Lineberger 1988, Brand & Lineberger 1992a), Juglans (Brand & Lineberger 1992a), Prunus, Eucalyptus gunnii, E. citriodora, Pinus pinaster and Salix babylonica (Franclet et al. 1987). The level of juvenility improves with subculture up to a point, and the use of short culture intervals is critical (Franclet et al. 1987).
The early work led to much enthusiasm, but, as pointed out by Timmis & Ritchie (1988), was based frequently on a very limited range of genotypes, and little monitoring of post-culture behaviour. In many cases the rejuvenation has been only partial, e.g. for Sequoia sempervirens (Franclet & Franclet-Mirvaux 1992) and Betula papyrifera (Struve & Lineberger 1988, Brand & Lineberger 1992a). More recent reports suggest that the expression of juvenile features by mature explants is dependent on the continued presence of the culture conditions, and that post-culture reversion to a more mature state is common, e.g. for Betula papyrifera (Brand & Lineberger 1992a), Pseudotsuga menziesii (Timmis & Ritchie 1988, Timmis et al. 1992) and Sequoia sempervirens (Franclet et al. 1987).
Rejuvenation through in vitro micrografting, generally sequential, of adult scions onto juvenile rootstocks has been reported also for several species, e.g. Sequoia sempervirens (Franclet 1983, Huang, Suwenza et al. 1992), Sequoiadendron giganteum (Monteuuis 1986), Thuja plicata (Misson & Giot-Wirgot 1985), Pinus pinaster (Monteuuis & Dumas 1992) and Pseudotsuga menziesii (Pullman & Timmis 1992). In citrus cultivars, rejuvenation was progressive with sequential micrograftings, but only to the seventh transfer, and then only partial (Huang, Hsiao et al. 1992). As above, there is little evidence that complete and persistent rejuvenation can be achieved through micrografting in vitro.
Paradoxically, there are several reports also of in vitro culture causing an accelerated maturation of juvenile explants. Trees micropropagated from juvenile tissues of several fruit tree species have flowered precociously (Zimmerman et al. 1985). Evidence of accelerated maturation with respect to morphological traits has been recorded for Pinus pinaster (Monteuuis & Dumas 1992), P. radiata, P. taeda and Pseudotsuga menziesii (Timmis & Ritchie 1988).
The rapid advances in techniques for molecular analysis has led to an increasing emphasis on studies of maturation at the molecular level. Hutchison & Greenwood (1991) concluded that the stability and reversibility criteria for maturation could be consistent with regulatory mechanisms based on DNA methylation, alterations in chromatin structure, and cellular mechanisms such as the internalization and turnover of membrane-bound receptors. These authors cite maize work where a particular transposable element is more actively expressed in the juvenile than in the mature portion of the plant, and the level of expression is inversely correlated with the level of DNA methylation in the transposable element regions. Maturation effects in Larix, however, are not obviously associated with differences in levels of DNA methylation (Hutchison & Greenwood 1992).
Recent work has been directed towards detecting genes which are differentially expressed in juvenile and mature trees. A protein, of unknown function, is associated with juvenility in a clone of Sequoiadendron giganteum (Bon 1988), and several proteins in Betula papyrifera (Brand & Lineberger 1992b). A few identified genes have been shown to be differentially expressed. For Larix laricina, the use of gene-specific probes has shown that three loci encoding chlorophyll a/b (cab) binding proteins are differentially expressed regardless of genetic background, while another two are differentially expressed in some backgrounds (Hutchison & Greenwood 1992). This suggested to the authors that maturation-related control of cab gene expression is operating through more than one element. Work with Pinus strobus is aimed at the isolation of root initiation specific genes by differential screening and subtractive hybridization of cDNA clones prepared from RNA purified from hormone treated cuttings (Goldfarb, Hackett et al. 1992). In the traditional ivy model, the absence of anthocyanin accumulation in mature plants is due to the absence of expression of dihydroflavonol reductase (dfr), and regulation is known to be at the transcription level (Hackett 1992). The cloned ivy dfr gene is to be used in in situ hybridization experiments aimed at cellular localization of mRNA for dfr. Such in situ hybridization experiments may determine whether genes for the different phase related characteristics are turned on or off at one time (one switch) or different times (several switches) during the maturation process (Hackett 1992). A factor complicating this work is that maturation changes are generally quantitative rather than qualitative, and it is therefore alterations in level of gene expression, rather than presence or absence, which are to be detected (Hutchison & Greenwood 1991). Separation of causes and effects of maturation presents a difficulty in interpretation of data (Greenwood 1992).
To summarize, there is little evidence that the maturation state can be reliably and adequately manipulated by in vitro culture, and further empirical work with this objective is likely to have a low probability of success. An understanding of the molecular basis of maturation is much more likely to lead to practical manipulation, but this work is in its infancy, and acceleration or reversal of maturation to precise levels remains a distant prospect.
Major practical implications of the maturation state for commercial forestry are related to:
There are a few forest tree species, e.g. poplars, Cryptomeria, and some eucalypts, for which the commercial planting of characterized clones is a reality. For most conifers, however, the propagation of superior full-sib families remains the most demonstrably achievable approach to maximizing the capture of genetic gains made in advanced breeding programmes. A well- documented limitation to the wider use of clonal forestry is the requirement that propagules display an acceptable level of juvenility with respect to ease of propagation and to subsequent field performance. For many forest tree species, this level of juvenility is not demonstrated by propagules from trees of selectable age. Manipulation of the maturation state, with respect to these important criteria at least, is therefore critical to the capture of gains through clonal forestry. Two alternative approaches to the problem are:
“Rejuvenation” - the derivation of juvenile propagules from trees of selection age, followed by multiplication of the selections. For some trees, for example many species of Eucalyptus and Araucaria, the stimulation of the development of coppice shoots from juvenile meristems which persist near the base of the stem is the simplest approach. Persistent basal meristems are not characteristic, however, of most tree species.
Maintenance of juvenility for the period of a clonal test, followed by multiplication of the best clones. Alternative approaches to the maintenance of juvenility include long-term hedging (Libby, Brown and Fielding 1972) and perhaps in vitro storage as discussed in Chapter 3.
The latter approach is perhaps less challenging physiologically and offers the advantage of increased heritability (clonal mean rather than individual tree), but involves the added time and expense of clonal testing. Other important parameters to be considered are:
The response rate - additional selection is imposed to the extent that individual genotypes do not display a satisfactory level of response to the treatment.
The time involved in multiplication of the selections.
These factors have raised some doubts regarding the value of clonal forestry, particularly in situations where the production of full-sib families is quick and easy.
The value of rejuvenation and maintenance of juvenility in clonal forestry are compared in Figure 12.1. This constitutes a comparison of propagation strategies for the hybrid between slash and Caribbean pines, quantified and plotted in the manner described in Chapter 11. Options compared in Figure 12.1 are:
This analysis assumes the availability of a rapid multiplication procedure for the rejuvenation options. Rapid multiplication is not as critical for the maintenance of juvenility option, because multiplication could be commenced before final results of the clonal test were available. This analysis is based on many assumptions concerning factors such as population size, genetic parameters, number of clones tested, and number of genotypes propagated. Sensitivity to these factors has been tested (data not presented here), and conclusions are:
Compared to the propagation of families, propagation of clones selected purely phenotypically is not particularly attractive, unless heritability is quite high - higher than that prevailing generally for growth traits.
Propagation of selections made on the basis of an index incorporating family information is considerably better.
For a clonal test of reasonable size, the propagation of tested clones for which juvenility has been maintained is about as good or better than rejuvenation of index selections. This conclusion will be different for traits of very high heritability.
The general conclusion then is that, where clonal testing is feasible and affordable, maintenance of juvenility is about as useful as rejuvenation for the capture of gains through clonal forestry. Strategies incorporating the maintenance of juvenility are appropriate then for most of the major industrial species. The ability to rejuvenate selections made on the basis of an index would undoubtedly confer added flexibility, but would only be really beneficial where heritability was high, and/or where clonal testing was difficult. This might apply to selections for some wood quality traits, and also as an interim measure for long rotation species, while results of lengthy clonal tests are awaited.
The propagation option combining phenotypic selection, rejuvenation and then clonal testing is not included in Figure 12.1, but it can be noted that, under most conditions, the time penalty of running two consecutive tests is not compensated for by the additional gain achieved through the preliminary phenotypic screening.
The generation interval comprises the selection interval (time from planting of a progeny trial to the point at which superior genotypes can be identified) and what could be termed the “recombination interval” (comprising, for example, time to graft selections, waiting for grafts to flower, development of seed following pollinations, and a nursery phase prior to the planting out of seedlings of the next generation). For a breeding program of a given size, the annual rate of gain will be inversely proportional to the length of the generation interval. Very large reductions in the generation interval will result in very large increases in the rate of improvement, and furthermore could facilitate the use of breeding strategies not commonly used for forest tree species, e.g. incorporating more inbreeding. (Inbreeding generally results in growth depression in forest trees, but nevertheless could conceivably be of value as a component of a breeding strategy - as it has been for crop species such as maize). The greatest value of reduction of the generation interval will be for long rotation species. Several techniques have demonstrated efficacy in the promotion of flowering (Bonnet-Masimbert 1987), and significant reductions in the generation interval are being achieved in some tree improvement programmes through the application of these (together with early selection methods). Major reductions, however, will require the availability of much more effective maturation control measures.
For clonal forestry with industrial species, maintenance of juvenility is about as useful as rejuvenation for many purposes, and probably achievable using technologies such as cryopreservation or coppicing. Nevertheless, more fundamental control of the maturation state remains one of the most valuable objectives of long-term strategic research in forest tree improvement. The ability to advance (in particular) and reverse maturation would confer large gains, but, for best advantage, must be combined with techniques for early selection. Rejuvenation is most applicable to programmes where good breeding programmes are in place and clonal forestry is a realistic goal. In particular, this applies to some programmes with conifers. As noted elsewhere, the need is not so urgent for the eucalypts because rejuvenation can be achieved by other means. Effective rejuvenation ultimately may have some application to other tropical hardwoods, as good breeding programmes are established for these. Maturation is a much less important issue with many of the non-industrials. Manipulation of the maturation state to induce early flowering and reduce generation intervals is potentially of greater interest, for industrial species at least, but only of real value where active breeding programmes are in place.
There is little evidence that the required control will be achieved by further empirical research with in vitro manipulation, and such work is difficult to justify, particularly if conducted at the expense of other activities. A capacity for precise manipulation of the maturation state is more likely to eventuate from an understanding of the underlying molecular processes, and this should be the primary emphasis of maturation research, probably best conducted with a few appropriate model species. Such work, however is in its infancy, and a clear understanding of the processes is probably many years away.
Figure 12.1 Comparison of clonal forestry options for Slash × Caribbean pine