Visual observations regarding somaclonal variation and reinfection with leafroll

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Visual observations regarding somaclonal variation and reinfection with leafroll in established Roobernet vines following plant material improvement by somatic embryogenesis

Piet Goussard

PG Goussard, Viticulture and Oenology Department, Stellenbosch University, Private Bag X1, Matieland, 7602
Key words: Somatic embryogenesis, somaclonal variation, leafroll, reinfection, distribution.
Introduction
Spontaneous somatic embryogenesis was established as a successfully repeatable technique in various grapevine cultivars, including Cabernet Sauvignon, Cinsaut noir, Gewürztraminer, Roobernet, Shiraz, Sultanina as well as the rootstock cultivar Rupestris du Lot, by the Viticulture and Oenology Department during the period 1987 - 1990. Several researchers (Krul & Worley, 1977; Mullins, 1987; Bouquet, 1989; Martinez et al., 1997 and others) have emphasised, however, that the above-mentioned technique, regardless of how successfully it is conducted, is not recommended for large scale plant propagation. These communications are based on the fact that regenerated embryos are not selected or synchronised and may therefore result in somaclonal variation. Somaclonal types of variation may range from visually easy to detect (e.g. deviations regarding leaf architecture and general morphological vine characteristics) to less obvious (e.g. subtle deviations in vigour, bunch and berry sizes, sugar and acid concentrations, flavour components, time of ripening, disease resistance, etc).

Fig 1. Source vine of Roobernet, visually heavily infected with leafroll.

Fig 2. Representative leaf and bunch examples of the source vine in Fig 1.

Fig 3. Proliferating embryos.

An important consequence of this technique, after the discovery by Namba et al. (1979) that clostero viruses (as associated with leafroll in grapevine) are limited to adult vascular tissue (phloem), was that somatic embryogenesis may be utilised as powerful and successful technology with regard to the elimination of clostero viruses from grapevine propagation material. Detailed studies on the ontogeny of somatic embryos confirmed the absence of vascular tissue in embryogenic callus, as well as the absence of vascular tissue compounds between regenerated embryos or between embryos and the mother tissue (ex plants) (Newton & Goussard, 1990). Against this background the effectiveness of somatic embryogenesis was demonstrated with regard to the overall and successful elimination of phloem bound viruses from infected propagation material (Goussard et al., 1991).
As virus elimination does not in any way contribute to the build-up of resistance in regenerated material, reinfection of vines (after establishment as material that is free from harmful viruses) and subsequent distribution is a well-known and serious problem in the SA grapevine industry (Carstens, 2001). Additionally it should be borne in mind that at some stage or another, propagation material ex somatic embryogenesis must be multiplied for the establishment of vineyards, which might again result in the possibility of somaclonal variation. Against this background the purpose of the present investigation was to characterise established vines of Roobernet (after subjecting leafroll infected material to spontaneous somatic embryogenesis) visually over a 12 year period with regard to two specific categories, namely (i) the occurrence and course of possible somaclonal variation and (ii) when and in which vines the first indications of reinfection with leafroll could be observed.
Material and methods
A source vine of Roobernet (Figures 1 & 2) was selected based on severe visual infection with leafroll and material thereof was subjected to somatic embryogenesis in 1992. From proliferating embryos (Fig 3) 10 adults were randomly collected and after germination, each was multiplied to five individual plants that were cultivated on their own roots in a clima room. Serological tests and electronmicroscopic investigations confirmed that regenerated plants were free of the viruses with which the mother material had been infected. After lignification, winter cane sections were cold stored according to standard procedures for future aerial grafting in the ampelographical collection at Welgevallen experiment farm.
The collection in question was established in 1991 using certified material of Chenin blanc/Richter 99 and is situated with a visually severely leafroll infected "Gloryvine" hedge on the northern and western borders. Vine spacing amounts to 2.7 m x 1.2 m, the vineyard is trellised (seven strand hedge with moveable foliage wires) with an east-west row direction and 30 vines per row. Since establishment the vineyard has been subjected to normal practices, but has not received any supplementary irrigation.

Fig 4. After 12 years this Roobernet row reflects complete evenness i t o vine size. Red to reddish brown leaves are due mainly to downy mildew and mechanical damage (topping machine). Photo: April 2006.

Fig 5. Smaller leaves (right) alternate with normal leaves (left) on main shoots.
From 1993 the collection was developed by top grafting of the existing Chenin blanc with certified material from alternative cultivars (three vines per cultivar) to comply with its status as field laboratory for student training. In November 1994 one entire row of 30 vines was top grafted to Roobernet by means of aerial grafting using material ex somatic embryogenesis in such a way that each of the 10 original embryos found representation in three vines. The vines were characterised visually on an annual basis and grapes were subjected to determinations of sugar concentrations. Due to the limited number of vines and the use of the collection for student training, no statistical analyses and interpretations of relevant data could be undertaken.
Results
Vigour and growth
Following an excellent take percentage (100%), vines showed strong vigour with upright growth (which is characteristic of Roobernet). After 12 years the particular vine row is still absolutely even with no visual occurrence of variation i t o vine size (Fig 4).
Morphological characteristics

Leaf characteristics
Based on ampelographic descriptions, no changes or variation could be observed in 27 of the 30 vines. The remaining three adjacent vines (in the middle of the row and deriving from an individual embryo) had leaves that were smaller with very obvious rectilinear lateral sinus fronts on the main shoots (Fig 5). It is interesting that in all three instances smaller leaves did not occur throughout, but were interspersed with normal leaf sizes without any other deviations being noticed in shoot characteristics. This situation has improved in the course of the cultivation period up to now (2006), but is still noticeable visually.

Fig 6. Since establishment, vines have performed optimally, with regular production of bunches that display the typical characteristics of Roobernet. Photo: March 2006.

Bunch and berry characteristics
There was no visually noticeable variation in bunch sizes and forms nor in berry sizes and forms, including vines with smaller leaves. Since establishment the vines have performed optimally, with regular production of bunches, in line with typical Roobernet characteristics (Fig 6).
Reinfection with leafroll
Despite regular and concerted efforts to control ants and mealybugs in the collection effectively, this goal could not be entirely achieved during the cultivation period from 1991 - 2006. During the autumn of 1998 the first visual signs of reinfection with leafroll were observed in three Pontac vines (row 1, adjacent to the leafroll infected "Gloryvine" hedge). In the period 1998 - 2006 leafroll spread visually in that particular row to neighbouring vines of Cabernet Sauvignon and Souzao, with an accompanying increase in intensity of visual symptom expression. Although not quite as severe (slow tempo), visual reinfection also occurred over the top of rows, from north to south. With the exception of the above-mentioned Pontac, Cabernet Sauvignon and Souzao, vines in the total collection of 12 rows, despite increasing visual reinfection, are still looking fairly "healthy" as far as leafroll is concerned, without serious effects i t o vine sizes and general performance.
The above-mentioned situation also applies to the Roobernet vines in question (row 5). In 2003 the first signs of reinfection were noticed in three non-adjacent vines in the middle of the row. These observations were based on the occurrence of regular chocolate brown discolourations over the entire leaf surfaces. Although the above-mentioned reinfection spread to eight vines in the particular row over the past three years, visual leaf discolourations did not increase significantly in intensity. Currently (2006) vines are still visually free of characteristic and severe leafroll symptoms (chocolate brown, brittle leaves with green veins and downward curl of leaf edges) and are still mostly bedecked in normal autumn colours (yellow-orange-light red). Visually there are no obviously smaller bunches with poor colouration in correctly positioned bearer shoots.

Fig 7. The occurrence of dark red to reddish brown leaves that occur up to two bearer positions on this vine. Note the small and shrivelled bunches - the result of wood rot ('tandpynagtig') and dead arm disease-like.
What is noticeable, however, is the occurrence (2006) of dark red to reddish brown leaves on main shoots in two non-adjacent vines. This phenomenon is not representative of the entire vine(s), but limited to individual bearer positions on the cordon(s) (Fig 7). A close association of the above with visual observations of wood rot ("tandpyn") and dead arm disease-like is applicable.
Sugar concentration and berry colour
Over the cultivation period (1994 - 2006) an average sugar concentration of 22.4øB was maintained. In addition bunches displayed excellent colour formation (including vines that were visually infected with leafroll).
Discussion and conclusions
Somaclonal variation
The occurrence of smaller leaves in vines deriving from an individual embryo may be ascribed, according to the literature (Krul & Worley, 1977; Mullins, 1987; Bouquet, 1989; Grenan, 1992; Martinez et al., 1977 and others), either to somaclonal variation or to a juvenile phase which will eventually pass. In this particular investigation it is difficult to make a proper distinction, especially since observation relies on a visual basis only. Even so, although this variation was extremely slight without any obvious detrimental effects on in-vineyard performance of vines, observations suggest that the possibility of somaclonal variation emanating from spontaneous somatic embryogenesis should be taken into account. It is therefore important that situations such as these be effectively controlled and managed as soon as any signs thereof are noticed, specifically in instances where large scale plant propagation is to be undertaken via somatic embryogenesis.
Reinfection with leafroll
Visual observations indicate that from a phytosanitary point of view, the quality of the original vines (Chenin blanc/Richter 99) is high (free of harmful viruses). It is well known that if vines of dubious phytosanitary quality are top grafted to alternative cultivars, leafroll (if present) is immediately transferred to the grafted certified component and may be visually observed as such in red wine cultivars, especially during the first growing season. In this investigation using Roobernet it was not the case and it may be stated almost unequivocally that the entry of leafroll should be ascribed to reinfection at a later stage, due to mealybugs as vectors.
Since normal yellow-orange-light red autumn colours are associated with Roobernet, special care should be taken i t o visual observations regarding the presence or absence of leafroll. Red and even brownish red discolourations may easily be confused with potassium and magnesium deficits, wood rot such as dead arm disease, strangling effects from ropes and even downy mildew infection on younger leaves late in the season. To make sure, the material in question of both Roobernet and "Gloryvine" should be subjected to specific virus detection tests.
Compared to a previous case study (Goussard & Underhay, 2004) with regard to the distribution of leafroll and characterisation of infected vines (Cabernet Sauvignon/Richter 99), the leafroll infected Roobernet vines in this investigation should be classified as visually lightly infected. Given the extremely slow distribution, accompanied by a very low intensity of symptom expression over the past four years, the vines in question should be able to operate productively for a reasonable period without sacrificing quality and quantity. At this stage wood rot and possibly dead arm disease pose a bigger threat as regards degeneration - especially if this should occur in conjunction with leafroll.
Summary
Despite the fact that all observations occurred on a visual basis and that this investigation is therefore not based on an in-depth scientific study, interesting observations regarding the occurrence and course of somaclonal variation, as well as reinfection with leafroll, could be made in trial vines over a cultivation period of 12 years. After all, a row of vines like this also presents one with the opportunity to select "positive" variations/deviations (should they occur) to be included in clonal development, should such a need present itself.
References
Bouquet, A., 1989. Culture in vitro de la vigne. Attention aux mauvaises surprises. Progr. Agric. Vitic. 106, 303 - 305.
Carstens, R., 2001. Rolblaar oorsig. LNR Infruitec-Nietvoorbij/Winetech bladskrif. 8p.
Goussard, P.G., Wiid, J. & Kasdorf, G.G.F., 1991. The effectiveness of in vitro somatic embryogenesis in eliminating fanleaf virus and leafroll associated viruses from grapevines. S. Afr. J. Enol. Vitic. 12, 77 - 81.
Goussard, P.G. & Underhay, J.P., 2004. Rolblaarverspreiding en karakterisering van geïnfekteerde wingerdstokke (Cabernet Sauvignon/Richter 99) te Welgevallen proefplaas - 'n Gevallestudie. Wynboer Tegnies 176, 84 - 87.
Grenan, S., 1992. Micropropagation of grapevine (Vitis vinifera L.). In: Bajaj, Y.P.S. (ed). Biotechnology in Agriculture and Forestry: High Tech and Micropropagation, Springer-Verlag, Berlin, pp. 371 - 398.
Krul, W.R. & Worley, J.F., 1977. Formation of adventitious embryos in callus cultures of "Seyval", a French hybrid grape. J. Amer. Soc. Hort. Sci. 102, 360 - 363.
Martinez, M.C., Boursiquot, J-M., Grenan, S. & Boidron, R., 1997. Etude ampélométrique de feuilles adults de somaclones du cv Grenache noir (Vitis vinifera L.). Can. J. Bot. 75, 333 - 345.
Mullins, M.G., 1987. Propagation and genetic improvement of temperate fruits: the role of tissue culture. In: Green, C.E., Somers, D.A., Hackett, W.P. & Biesboer, D.D. (eds). Plant Tissue and Cell Culture, Liss, Inc., New York, pp. 395 - 406.
Namba, S., Yamashita, S., Doi, Y., Yora, K., Terai, Y. & Yano, R., 1979. Grapevine leafroll virus, a possible member of closteroviruses. Ann. Phytopath. Soc. Japan 45, 497 - 502.
Newton, D.J. & Goussard, P.G., 1990. The ontogeny of somatic embryos from in vitro cultured grapevine anthers. S. Afr. J. Enol. Vitic. 11, 70 - 75.
Summary
Visual observations were carried out on the occurrence of somaclonal variation as well as reinfection with leafroll associated viruses within the cultivar Roobernet of which material was subjected to spontaneous somatic embryogenesis for virus sanitation. Somaclonal variation, characterised by smaller leaves, was observed in three out of a total of 30 established vines. Although eight vines appeared visually reinfected with leafroll after 12 years, the spread thereof and intensity of symptom expression were limited, despite the presence of severe numbers of mealybugs in the ampelographic collection. However, the occurrence of somaclonal variation within material regenerated by means of spontaneous somatic embryogenesis clearly shows the need to control it efficiently, especially in cases of mass propagation procedures.
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