Juvenile Rootstocks Research Articles

Controlling the barriers of cloning mature Picea abies (L.) H. Karst. via tissue culture and co-cultivation with Agrobacterium rhizogenes

Providing an improved protocol for cloning of Picea abies (L.) H. Karst. mature trees via Agrobacterium rhizogenes. In reforestation programs, rejuvenation of superior genotypes is of great importance in helping juvenile rootstock production. In this regard, the use of plant tissue culture to produce axillary and/or adventitious shoots from explants of mature trees has been the objective of many researchers. There are a few reports about the successful in vitro rooting of the regenerated shoots from explants of mature trees. There is a very low rate of natural rooting in conifers, between 1 and 5%. Furthermore, they are still hard to root even when auxins are applied. In this study, in vitro propagation of Norway spruce explants and its rooting improvement by A. rhizogenes with the aim of receiving an efficient clonal propagation method was investigated. The effects of BA (0.5, 1, 1.5, and 2 μM) and TDZ (0.0025, 0.025, 0.25, and 2.5 μM) were examined on axillary shoot development from shoot-tip explants of the Norway spruce. The effects of three strains of Agrobacterium rhizogenes (K599, AR15834 and GMI9534) were also examined on enhancement of the root induction. According to the results, after the treatment of 1.5 μM BA, 87.5% of shoot-tip explants produced axillary shoots (the highest percentage among treatments), and also the maximum mean number of regenerated shoots per explant (6.5) was achieved in this treatment. Furthermore, the induction of rooting was performed using IBA (7.5 µM) and IAA (0.5 or 1 μM) in the WPM medium along with keeping the explants in the darkness at 21 °C, which yielded 37.5% and 12.5% rooting, respectively. Moreover, the application of coumarin along with IAA caused an increase of 22% in rooting, compared to the use of IAA alone. Two strains, K599 and AR15834, succeeded in improving the percentage of rooting, with the highest being 54.2% as a result of using the K599 strain alongside 7.5 μM IBA. The proposed treatment can be used efficiently for the propagation of Norway spruce in landscaping and/or reforestation programs.

Successive grafting confers juvenility traits to adult Spanish red cedar (Cedrela odorata Linnaeus): a tool for the rescue of selected materials

Spanish red cedar, Cedrela odorata L. (Meliaceae), is a valuable timber tree in tropical American forests. Existing demand for elite individuals endangers the conservation of interesting germplasm, prompting the development of efficient protocols for the establishment of orchards for tree breeding. Based on previous work regarding grafting adult individuals onto young rootstocks, superior trees from Calakmul Biosphere Reserve in Campeche, Mexico were employed as a source of explants for plant regeneration after successive rounds of grafting and micrografting onto juvenile rootstocks. The results showed that root length and appearance, internode distance, leaf length and number, and plant height values for trees derived from successive rounds of grafting and micrografting were similar to those obtained from juvenile trees derived from seeds, suggesting that developmental traits associated with reinvigoration were partially induced following the reported procedure. This protocol may be useful for the propagation of mature elite trees belonging to the Meliaceae family.

Increased endogenous indole-3-acetic acid:abscisic acid ratio is a reliable marker of Pinus massoniana rejuvenation

ABSTRACTPinus massoniana is a recalcitrant tree species for rooting in vitro. We rejuvenated 26-year-old P. massoniana trees by successive grafting. Rooting rates of rejuvenated shoots were > 83.1% after rooting induction. We compared endogenous levels of indole-3-acetic acid (IAA), abscisic acid (ABA), gibberellins (GAs) and zeatin-riboside (ZR), and the rhizogenesis ability of axillary shoots of mature and rejuvenated materials in vitro, i.e., somaplants and grafts. Enhancement of the rooting ability of mature materials in vitro following somatic embryogenesis or repeated grafting onto juvenile rootstocks was accompanied by increased IAA and GAs levels, and by decreased ABA levels in scions used as starting material for micropropagation in vitro. Successive subcultures did not influence the rooting ability of shoots from untreated mature material. Rooting ability of shoots in vitro, however, gradually increased with subculture frequency during repeated subculturing in grafting materials. The IAA:ABA ratio in shoots in vitro after grafting five times, and consequently capable of root organogenesis, was higher than in shoots of untreated mature material incapable of root organogenesis in vitro. A high IAA:ABA ratio was detected in scions of somaplants that were capable of rooting in vitro despite subculture times. We found that the endogenous IAA:ABA ratio is a reliable marker for the recovery of root organogenesis in vitro after rejuvenating treatments for mature P. massoniana trees.

No signs of meristem senescence in old Scots pine

Summary Ageing and senescence in plants remain poorly understood. Although meristem totipotency may allow woody perennials to be immortal, relative growth and photosynthetic rates typically decline with age. Trees of ages between 129 and 534 years were selected in one of the oldest extant populations of Scots pine. Apical branches were propagated by grafting onto homogeneous juvenile rootstock to eliminate the effects of size and environmental variability and isolate those due to age. The hormonal profile of leaves and seeds along with markers of the physiological status of leaves and their pattern of DNA cytosine methylation were measured 15 years after grafting. The percentage of total methylated loci in nuclear DNA increased with increasing meristematic age. However, only very few significant relationships were found between levels of phyto‐hormones, pigments or physiological markers either in leaves or seeds and age of the meristem. In addition, shoots grafted from old trees grew as fast as those from younger trees and produced the same number of germinable seeds. Synthesis. We conclude that changes in DNA methylation can occur in old trees. The lack of apparent physiological deterioration in the grafted plants suggests that meristem senescence is not the main factor triggering whole‐plant ageing in Scots pine.

Regulation of Foliar Plasticity in Conifers: Developmental and Environmental Factors

Foliar plasticity is widespread among woody plants, and historically most observations have been made in angiosperms. This review concentrates on examples from gymnosperms, particularly the Pinaceae, since there have been a number of recent studies on coniferous forest species. Foliar plasticity can be defined as variation in the morphology and physiological function of foliage produced over time and space within a single individual. For example, leaves produced in the sun have a higher leaf mass area (LMA) than leaves produced in the shade, and differ in their photosynthetic capacities. Sun and shade leaves are examples of heterophylly, or variation in leaf morphology in response to environmental variation in the immediate space surrounding the apical meristem producing the leaf primordia. The LMA of coniferous foliage also tends to increase with age, independently of the external environment, and this variation, called heteroblasty, appears to be a result of maturation of the apical meristem, which occurs over time. The regulation of variation due to heterophylly and heteroblasty appears to be very different. Heterophylly in response to light appears to vary linearly with available light. More massive sun foliage produced by a given apical meristem may be a response to available Photosynthetically Active Radiation (PAR) and its effects on net photosynthesis, or may be a photomorphogenetic response to the ratio of red to far red light. In contrast, heteroblastic variation can occur independently of available light, and may be the result of the developmental age of the apical meristem. It appears to vary curvilinearly with age, with the most rapid change occurring during the earliest life stages of the plant. Heteroblastic variation appears to be less plastic than heterophyllic, since grafted scions from mature or juvenile donors retain LMA characteristics of donor whether grafted onto juvenile or mature rootstocks.

Production of transgenic adult plants from clementine mandarin by enhancing cell competence for transformation and regeneration

Genetic transformation of mature trees is difficult because adult tissues are recalcitrant to Agrobacterium tumefaciens infection and transformation and because transgenic mature events are less competent for regeneration. We have shown that reinvigoration allows manipulation of the vegetative phase to increase the potential for transformation and regeneration without loss of competence for flowering and fruiting. To produce transgenic plants from clementine mandarin (Citrus clementina hort. ex Tanaka), we optimized the conditions of the source material both ex vitro and in vitro. Grafting of mature buds on juvenile rootstocks in the spring and preventing multiple bud sprouting by removing all but one bud permitted selection of vigorous first flushes for in vitro culture. Use of additional virulence genes from A. tumefaciens to increase transformation frequency and optimization of culture media and conditions to enhance explant cell competence for T-DNA integration and organogenesis resulted in efficient and reliable transgenic plant production. Transformed regenerants from explants, cultured in media without antibiotics, were identified by a screenable marker (either beta-glucuronidase or green fluorescent protein (GFP)), creating the possibility of generating transgenic clementine plants without antibiotic resistance marker genes. Stable integration of foreign genes was demonstrated by Southern blot analysis, and expression of these foreign genes was confirmed by detection of GFP fluorescence in leaves, floral organs and fruits of the transgenic plants.

Effects of serial grafting, ontogeny, and genotype on rooting ofQuercus rubracuttings

Bud grafts, up to three series over 3 years, were made on seedling and tree rootstocks using scions from juvenile and mature northern red oak (Quercus rubra L.). Serial grafts on juvenile rootstock used buds collected from shoots developed from grafted scions of prior years. Rooting trials were performed in years 2 and 3 with shoot cuttings developed in situ on seedlings and trees and developed from successful grafts. Without grafting, cuttings from seedlings rooted more frequently and had more roots than cuttings from trees. Significant variation within maturation groups due to genotype and ontogeny obscured absolute between-group differences. Grafting scions of juvenile origins onto seedling rootstock had little effect on percent rooting and the number of roots for cuttings. Grafting onto seedling rootstock tended to increase rooting and the number of roots for cuttings from mature origins, but the effect was not progressive with increasing grafting series. Grafting onto mature rootstock did not affect rooting of cuttings from juvenile or mature origins collected in the first growing season after grafting, but cuttings from juvenile scions collected in the second growing season exhibited reduced percent rooting compared with cuttings from seedling controls. Results suggest that northern red oak buds are predetermined in their developmental fate relative to rooting parameters and are only minimally influenced by grafting. The true effect of grafting on the subsequent rooting of cuttings may be mediated through processes other than rejuvenation.

Growing Olives in Texas

Olives have not been commercially grown in Texas because earlier investigators considered Texas climate inappropriate for olive cultivation. No experimental investigations were conducted in Texas. Olives, however, grow in very diverse climatic conditions throughout the world. More than 2000 varieties of olives have been reported worldwide. Olive accessions have also shown adaptability to local climates. United States imports thousands of tons of olive oil each year. Some of these requirements could be fulfilled internally if vast lands available for cultivation in Texas could be utilized for olive production. We have started experimental research growing olives in Texas. Our initial surveys of olives groves established during the last 4-6 years have revealed the existence of at least two trees with remarkable adaptability to Texas conditions. One of these trees had flowered and fruited in the Rio Grande Valley where earlier workers had predicted that flowering in olives will not occur. Thus, there appears enough genetic diversity and clonal variations even among existing olive trees in Texas that there is a hope for viable cultivations of olives in Texas. Clones from these trees have now been produced for testing at various locations within the Texas Valley. Grafting experiments have shown that scions from flowering shoots could produce flowers even on juvenile rootstocks thus providing quicker methods for propagating and evaluating selected clones. Relatively inexpensive chilling chambers were developed to study chilling requirements for flower induction in selected olive varieties. Out initial data shows that Arbequina could flower under mild chilling conditions.

In vitro Micrografting of Mature Pistachio (Pistacia vera var. Siirt)

The success of various in vitro micrografting methods of shoot tips of pistachio (Pistacia vera L. var. Siirt) have been examined. Excised zygotic embryos that germinated in vitro were used as rootstocks. Current year shoot tips from mature trees of pistachio micrografted onto in vitro juvenile rootstocks, resulted in the restoration of shoot-bud proliferation. Variables tested include a size of microscion, grafting method, effects of culture medium and effects of time of the year at which shoot tips were used. The results indicate that the easiest and most successful method for grafting was slit micrografting. High levels of micrograft take were achieved with 2–4 mm (56.75%) and 4–6 mm (79.25%) long scions obtained from the regenerated shoot tips. The survival rate of the shoot tips was directly related to time of the year. The best growth of microscion was obtained with the in vitro forced shoot tips rather than with shoot tips excised from tree. Slow growth and lack of axillary shoot development on the micrografts was noticeable when the micrografts were cultured on hormone-free and germination medium. In vitro micrografted plantlets were successfully weaned and no problems were encountered with the establishment of micrografted plants in vivo.

Manejo de plantas madres para el cultivo in vitro de segmentos nodales del guayabo (Psidium guajava L.).

Se cultivaron in vitro segmentos nodales de guayabo recolectados de brotes de injertos adultos sobre patrones juveniles, plántulas a plena exposición solar y bajo sombra (40%), plantas adultas sin y con sombra (80%). También se cultivaron explantes de plantas adultas cultivadas en el campo bajo las siguientes condiciones: control; 60 días después de la aplicación de dormex (cianamida hidrogenada 49%) al 2% y 4%; 60 días después de poda parcial; y 75 días bajo 80% de sombra. A los 60 días de cultivo, las plántulas y plantas adultas bajo sombra produjeron 0% de explantes oscurecidos. El máximo porcentaje de explantes con brotes de yemas (PBY) (100%) se obtuvo a los 20 días en explantes de plántulas, y a los 40 días en explantes de plantas adultas bajo sombra. A los 60 días, las plantas adultas e injertadas sin sombra produjeron 35% y 40% de explantes oscurecidos, respectivamente, y 85 y 80% de explantes con brotes de yemas, respectivamente. El control registró 65% de explantes brotados; los tratamientos con dormex al 2% y 4%, 80% y 70%, respectivamente; la poda parcial, 65%. La aplicación de dormex al 2% incrementó el PBY y el porcentaje de explantes con hojas formadas comparada con los del control.

Restoration of rooting competence in mature Faidherbia albida , a Sahelian leguminous tree, through serial root sucker micrografting

Faidherbia albida is an essential leguminous tree of arid areas of Africa which is widely used in agroforestry. This species naturally produces root suckers. Their morphogenetic ability has been used to initiate a rejuvenation/reinvigoration process in mature subjects. The proposed method contains two stages. The first stage generates the first ramets : our results have shown that suckers picked from root cuttings are more vigorous than explants taken directly from the crown of adult subjects. The second stage furthers the rejuvenation process through cycles of repeated micrografting onto juvenile rootstock, which have been grown in vitro. As early as the third cycle, a significant increase in reactivity is gained. This is determined by growth of the scion and the rooting competence of microcuttings severed from the scions after each micrografting cycle. At that point, the rooting competence is completely restored. So, this sequential method, (i) suckers from root cuttings, (ii) repeated micrografting, seems valid as a technique for the restoration of rooting competence in mature F. albida with a view to its cloning.

Age-related changes in foliar morphology and physiology in red spruce and their influence on declining photosynthetic rates and productivity with tree age.

The contribution of changes in meristem behavior to age-related decline in forest productivity is poorly understood. We studied age-related trends in needle morphology and gas exchange in a population of red spruce (Picea rubens Sarg.) growing in a multi-cohort stand where trees ranged from first-year germinants to trees over 150 years old, as well as in grafted scions from these trees. In the field study, age-related trends in foliar morphology were determined in six cohorts ranging in age from 2 to 120 years, and differences in gas exchange characteristics were compared between 60- and 120-year age classes. In a common-rootstock study, scions from trees representing 20-, 60-, and 120-year cohorts were grafted onto juvenile rootstock and maintained for three growing seasons, after which morphological and physiological foliar attributes were evaluated. The field study revealed significant age-related trends in foliar morphology, including decreasing specific leaf area, and increasing needle width, projected area, and width/length ratio. Similar trends were apparent in foliage from the grafted scions. Both in situ foliage and shoots of grafted scions from the oldest cohort showed significantly lower photosynthetic rates than their counterparts from younger trees; however, differences in stomatal conductance and internal CO(2) concentrations were not significant. These results suggest that: (1) foliage of red spruce exhibits age-related trends in both morphology and physiology; (2) age-related decreases in photosynthetic rates contribute to declining productivity in old red spruce; (3) declines in photosynthetic rates result from nonstomatal limitations; and (4) age-related changes in morphology and physiology are inherent in meristems and persist for at least 3 years in scions grafted to juvenile rootstock.

Reinvigoration of mature chestnut (Castanea sativa) by repeated graftings and micropropagation

Gradual reinvigoration of adult chestnut (Castanea sativa M. cv. Montemarano) shoots was obtained by serial grafting onto juvenile rootstocks. The phenomenon was evaluated on the basis of percentage of primary nodes regenerating axillary shoots and length and number of shoots (> 10 mm) per primary node. In vitro growth of explants from serially grafted shoots was significantly lower than that of explants from seedlings at the end of the establishment phase. Only microshoots from seedlings and plants that had been serially grafted four times could be subcultured on proliferation medium. Repeated subculture on medium containing a low cytokinin concentration induced progressive reinvigoration of microshoots derived from plants that had been serially grafted four times. The number of axillary shoots per explant increased significantly after six subcultures. After 12 subcultures, microshoots from serially grafted plants showed an increase in stem elongation, rooting and plantlet survival. After in vitro stabilization, there was no difference in in vitro performance between microshoots derived from seedlings and serially grafted plants. Microshoots multiplied from serially grafted plants displayed only a transitory appearance of juvenile traits.

Endogenous cytokinins as biochemical markers of rubber-tree (Hevea brasiliensis) clone rejuvenation

The endogenous levels of isopentenyladenine, isopentenyladenosine, zeatin and zeatin riboside and the ability forin vitro axillary shoot organogenesis and rhizogenesis were compared between mature and rejuvenated clones ofHevea brasiliensis (Mull. Arg.). Enhancement of thein vitro organogenesis ability of rubber-tree clones following somatic embryogenesis or repeated grafting onto juvenile rootstocks was accompanied by an increase of zeatin riboside levels in shoots used as starting material forin vitro micropropagation. Furthermore, the zeatin level, inin vitro shoots of clones treated byin vitro micrografting, and consequently capable of axillary shoot and root organogenesis, was higher than inin vitro shoots of non treated mature material incapable of in vitro organogenesis. We conclude that the endogenous zeatin-like cytokinin level (free and ribosylated forms) can be considered as a reliable marker for the recovery ofin vitro shoot and root organogenesis after rejuvenating treatments in rubber-tree clones.

Rejuvenation of Sequoia sempervirens in Vitro: Changes in Isoesterases and Isoperoxidases

Rejuvenation, as evidenced by restored rooting competence, of Sequoia sempervirens by a series of repeated grafting of adult shoot tips onto juvenile rootstocks in vitro was confirmed. Complete restoration of rooting competence was achieved after grafting repeatedly five times. The rejuvenation was correlated with a disappearance of adult-associated esterase and peroxidase isozymes and an appearance of isoesterases and isoperoxidases that were characteristic of juvenile-phase shoots. These esterase and peroxidase isozymes could serve as markers to assist phasechange investigations.

A protocol toward multiplication of the medicinal tree,Eucommia ulmoides oliver

A protocol based on shoot cultures of 1-mo.-old seedlings was developed for rapid asexual multiplication of eucommia, the source of an antihypertensive medicinal. The explant is an excised shoot tip, 3–5 mm tall. MS basal medium supplemented with 1 mg/liter BA is employed to establish primary cultures and subsequently multiply shoots. Shoots are subculturable on the same medium and can be increased at a rate of 7.5 new shoots per 2-shoot sector every 3 wk. Rooting is achieved in a Gelrite medium with the MS salts reduced to 1/3 strength and the BA replaced by 0.1 mg/liter NAA. The method is not directly applicable to mature trees. Applicability will require explants from rejuvenated sources, possibly attainable by the method of repeated grafting of shoot apices onto juvenile rootstocks, repeated subculturing of shoots, or culturing shoot apical meristems.

Phase reversal in Sequoia sempervirens in relation to mtDNA

Restriction fragment analysis revealed differences in mitochondrial DNA (mtDNA) between adult and juvenile coastal redwood, Sequoia sempervirens (D. Don) Endl. Thus, for example, juvenile shoots contained 4.0‐ and 3.6‐kb BamHI], restricted fragments that were absent in adult material. Rejuvenation by 5 repetitive graftings onto juvenile rootstocks resulted in the same 4.0‐ and 3.6‐kb fragments as were present in the juvenile material. The similarities in mtDNA fragments of juvenile and rejuvenated. shoots, as well as their distinction from adult shoots, were confirmed by double digestion, Southern hybridization and DNA sequencing.

Rejuvenation in vitro: Modulation of Protein Phosphorylation in Sequoia sempervirens

Summary Crude extract of whole cells and isolated mitochondria from cultured Sequoia sempervirens shoots of the adult, juvenile, and grafted adult tissues were incubated with [γ- 32 P]-ATP, and the resultant phosphoproteins were then separated by SDS-PAGE and examined through autoradiography. In the extracts of whole cells, the phosphorylation of their 32-kDa protein was evident only in the adult, and that of the 31-kDa protein was detected only in the juvenile cells. Repeated graftings of shoots from adult trees onto juvenile rootstock results in changing the phosphoprotein pattern. It becomes like that of the juvenile tissue and becomes very similar after four grafts. However, no qualitative differences were noted in mitochondrial phosphorylation patterns; rather, the total 32 P incorporation turned out to be lower in the adult tissue than in juvenile and rejuvenated tissues.

Factors controlling micropropagation of mature Fagus sylvatica

The influence of several endogenous factors on micropropagation of mature elite trees of beech (Fagus sylvatica L.) has been investigated. February was the most beneficial month for explantation of dormant buds, since infection with endogenous bacteria was still low and in vitro growth of the plant material was the highest. The genotypes tested gave different results concerning their growth on various tissue culture media. Out of 51 mature grafted genotypes only seven could be established in vitro. Grafting of branches of mature stock plants on juvenile rootstocks resulted in a high increase of multiplication rate compared with corresponding mature material, which could not be subcultured in vitro. Apical buds, larger than 20 mm, from 1-year-old shoots proved to be the most suitable explant source. Plant material could be subcultured for several years and rooted successfully.

Effects of number of scion leaves on inlay grafting in three durian clones.

In Malaysia, durian (Durio zibethinus Murr.) is traditionally grown from seed. As reported by FAMA (Federal Agricultural Marketing Authority), only about 3%of the durian grown in the state of Perak were budded I). Gradually, however, durian trees are vegetatively propagated, and the most common method presently practiced is bud-grafting. In recent years, many work and techniques have been done to offer growers propagation of durian2•3•6). Beside bud-grafting, cleft grafting is getting popular but there seems to be a tendency at the moment to use very juvenile rootstocks. With the exception of mangosteen and sapodiIIa, most fruits are using few weeks old Of course there are advantages in doing so, such as the ease of working, but there may be occassions where much older rootstocks need to be used. In the situation where the diameter of rootstock is much larger than that of the scion, grafting technique such as inlay grafting is suitable. Cleft grafting and bud-grafting are known to be good methods for young rootstock, but not for old rootstock. In the experiment, effects of inlay grafting for old rootstock was studied.