Plant Physiology 98(1): 166-173 (Jan. 1992)
Rejuvenation of Sequoia sempervirens by repeated grafting of shoot tips onto juvenile rootstocks in vitro
Li-Chun Huang, Suwenza Lius, Bau-Lian Huang, Toshio Murashige, El Fatih M. Mahdi, and Richard Van Gundy


Repeated grafting of 1.5-centimeter long shoot tips from an adult Sequoia sempervirens tree onto fresh, rooted juvenile stem cuttings in vitro resulted in progressive restoration of juvenile traits. After four successive grafts, stem cuttings of previously adult shoots rooted as well, branched as profusely, and grew with as much or more vigor as those of seedling shoots. Reassays disclosed retention for 3 years of rooting competence at similar levels as originally restored. Adventitious shoot formation was remanifested and callus development was depressed in stem segments from the repeatedly grafted adult. The reversion was associated with appearance and disappearance of distinctive leaf proteins. Neither gibberellic acid nor N6-beneyladenine as nutrient supplements duplicated the graft effects.


H. helix has been the classic model for phase-change investigations. But it may not be the best source of information for the broad range of plant species. Some fundamental findings, particularly the critical responses to giberellin, have not been duplicated with tree species. Gibberellin treatment has elicited some organ modifications, e.g. reappearance of juvenile leaves for a few cycles (4). But evidence of profound and lasting morphogenetic changes in the apical meristem has been lacking. Clearly, gibberellin treatment has not been very useful for exploring phase reversal of trees.

In contrast, rejuvenation has been more generally achievable when scions from mature plants were grafted onto juvenile rootstocks. In fact, the first evidence of reversion by grafting was demonstrated with H. helix (8). Nevertheless, whereas rejuvenation of trees occurs incrementally with successive grafts, that of H. helix is achieved after grafting only once. Progressive changes that are associated with the gradual shift in phases are, thus, not as easily observed in H. helix.

Grafting for rejuvenation can be performed in vitro (12, 13, 19) as well as in vivo (8, 9, 17, 24). Grafting in the former instance can be done with substantially smaller and more juvenile rootstocks; hence, regrafting can be performed at shorter intervals and significant levels of reversion can be attained more quickly. With S. sempervirens, regrafting is possible every 8 weeks and key juvenile traits can be observed after the four successive grafts. Furthermore, unless controlled- environment chambers are used in vivo, the in vitro procedure ensures more readily reproducible developmental conditions.

S. sempervirens was chosen because an earlier investigation had shown the ease with which cultures of continuously proliferating shoots could be established, both juvenile and adult, and had developed a rooting bioassay (25). Another study had disclosed differentiation of adventitious shoots in tissue culture (1).

The graft-associated rejuvenation of S. sempervirens is evident as progressive increases in incidence of rooting and number of roots, elongation and branching of stems, and vigor of roots and stems; restoration of juvenile leaves and competence for adventitious shoot differentiation; diminishment of callus growth; and reduction in mortality of explants. None of these changes are effected by nutrient medium addenda of gibberellin or cytokinin. The repression, especially of rooting, by GA3 and BA is not unexpected. Even at much lower dosages, both gibberellin and cytokinin have been established as inhibiting rooting in most species (15, 22). The rejuvenated states, based on increased rooting competence, were retained by stock-cultured shoots for more than 3 years, and at intensity levels that corresponded to the number of successive grafts. Unfortunately, none of the plants were established in soil to observe their further development, especially their cone-bearing characteristics.

Of considerable significance was the absence of some distinctive proteins and appearance of others in the leaves of rejuvenated S. sempervirens when compared with proteins from adult leaves. The two-dimensional PAGE patterns were essentially identical for repeatedly grafted adult and juvenile. Bon (3) has been able to correlate a 16 kD shoot apex protein with juvenility of Sequoiadendron giganteum, but the precise role of this protein remains unestablished. The protein observations, nevertheless, suggest a logical basis for studying phase change at the molecular level. Additional information now available includes the transmissibility of the signal through cells of the graft union; this probably implies a small sized molecule(s). Prolonged retention of restored traits, at least 3 years in vitro, suggests self-replicating molecule(s), perhaps nucleic acid(s). If it is nucleic acid, then it must be constructed to resist enzymatic destruction, e.g. as small circular DNA- or RNA-plasmids. The possibility of a new phytohormone is not excluded, inasmuch as Geneve et al. (10) recently reported on an unidentified graft transmissible factor in the lamina of H. helix, which over time promoted rooting in petiole of mature H. helix.

In view of the slight but significant reversion that occurs during prolonged culture of adult shoots, an observation consistent with other investigations (9, 26), protocols should be followed closely to obtain precise information.

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