Rapid propagation of grape planting stock
by M. Andrew Walker, Deborah A. Golino
Department of Viticulture & Enology, Foundation Plant Materials Service University of California, Davis, CA

A thriving international market for wine and grapes has resulted in an explosion in vineyard plantings in many parts of the world. One of the consequences in some countries has been a shortfall in desirable planting stocks. This is especially true for countries with strict quarantine regulations governing the importation of grapevines. In those countries, it is necessary for the domestic nursery industry to respond to demand for scarce varietal and clonal selections, or new rootstocks, with techniques that allow the rapid expansion of propagating stock.

Cirami and Ewart (1995) were referring to clonal evaluation in the following quote, but the statement is equally true for new varieties or rootstocks.6 "The identification of superior clones without a mechanism of vine certification and multiplication can lead to enormous frustration in the wine industry. The vine grower learns that improved planting material is available, he is just unable to obtain it."

Both the slow movement of new introductions through quarantine and certification programs and the relatively slow process of grape propagation via dormant cuttings are responsible for this frustration. There is great interest at this time in any technique that might speed up the process by which new materials are made available to growers. This is particularly true in California, where the current planting boom has prompted accellerated acceptance of some of the rapid propagation techniques.

California planting boom

The demand for grape planting materials is at an all time high in California. New vineyards are being planted in all areas of the state, and thousands of acres of vineyards once planted to the phylloxera-susceptible rootstock AXR1 continue to be replanted each year. Nurseries are struggling to keep up with demand.

The California Department of Food & Agriculture’s Agricultural Statistics Service tracks the grape acreage by variety and region. These records provide us with some statistics about the extent of the current planting boom, although records are reported for the previous year, and by some accounts (California Association of Winegrape Growers 1998), they may underestimate acreage of some varieties by as much as 40%.

According to these reports, four widely planted varieties have experienced remarkable growth in California (Figure 1). In 1982, there were 22,050 acres of Chardonnay, 21,289 acres of Zinfandel 16,640 acres of Cabernet Sauvignon, and 2,147 of Merlot. By 1991, there were 56,609, 34,369, 34,176, and 8,188 acres of each, respectively. The 1997 acreage records list 88,517, 50,498, 45,307, and 38,522. These four varieties now account for 54.7% of the 407,230 winegrape acres in California recorded for 1997.²

In addition to the increase in acreage of these major varieties, some varieties, which have previously been planted to a limited extent, are suddenly becoming more popular. For example, there is increasing interest in Sangiovese and Syrah. Prior to 1982, there were respectively 25 and 89 acres, of these varieties in California. By 1991 there were 232 and 413, and the acreage records for 1997 list 2,498 and 4,277. This represents a 50-fold increase in Syrah and a 100-fold increase in Sangiovese.

In addition to the demand for varietal grape materials from nurseries, the issue of clonal selection has become increasingly important to California growers and winemakers. It is not unknown in California for wineries to pay a premium for particular clones and to contract with growers based on the clones planted in selected vineyards. There is particular interest in clones from France and Italy, which are new to the U.S. These clones have been arriving in the state via legal and illegal channels and are now in great demand.

Although little data exist regarding how well these clones perform in California, they are being utilized in replanted vineyards and newly developed sites at an increasing rate. Several California nurseries are now collaborating with French and Italian nurseries, as well as vine improvement centers, such as ENTAV and Rauscedo, to provide California growers with better access to these clones. These joint ventures will increase the rate of new introduction, resulting in higher demand for these "new" products, and increased pressure on nurseries for rapid propagation.

The California table grape industry is especially quick to change to new varieties. The varietal loyalties of winemakers do not apply; successful new releases by breeders can become popular and even dominate the market in a very short period of time. Propagators need to respond to these table grape demands with very quick production schedules.

Economics and global demand for wine and fruit are fueling huge new plantings in California. But even when the demand levels off, there will still be a strong need for planting materials beyond this current generation of vineyard expansion.

For example, a majority of California’s vineyard acreage is currently planted without rootstock. These vineyards are on sandy soils in the Central Valley and are rarely affected by phylloxera. However, nematodes, most notably an array of root-knot species (Meloidogyne), build up to high levels on these soils, and the establishment of second generation vineyards on such sites will require use of nematode-resistant rootstocks.

Further, California is only in the early stages of regional evaluation of varieties, clones, and rootstocks. As new materials are tested, it can be anticipated that vineyards will need to be replaced to keep up with newly defined regional markets. Finally, new generations of rootstocks are being produced for California soils to resist specific pest complexes.¹⁹ The demand for these rootstocks will increase as sites are replanted to second and third generation vineyards.

Role of new propagating techniques

Traditional grape propagation techniques utilize mother vines, from which dormant cuttings are taken for rooting, bench grafting, or field budding. Mother vines are generally planted from dormant rootings or potted plants and require about three years to produce generous amounts of cuttings.

When new clones or varieties are released from breeders or from quarantine programs, there is very little wood available for distribution and a variety of techniques have been employed to produce commercial amounts of planting stock over a short period. Many of these techniques are labor and technology-intensive, and their cost may not compete with that of traditional methods of propagation when source materials are not limited. However, when planting stock of new varieties, clones, or rootstocks is limited, rapid propagation techniques can speed up release and offer large economic advantages.

The introduction of new planting materials through vine improvement associations is a major limitation to grape industry utilization of new clones, varieties, and rootstocks. Many of the rapid propagation techniques were developed by these associations to speed up the release of material and to avoid the spread or infection of certified material with pests and diseases.

Mist propagation of herbaceous cuttings

Rapid propagation techniques most often use herbaceous plant material ranging from apical meristems to partially lignified, 25 cm shoot cuttings. Propagation from herbaceous material must be done in very high humidities utilizing mist propagation or tissue culture techniques.⁹

The simplest procedure for the multiplication of an actively growing plant involves making multiple green cuttings and rooting them under mist propagation. Mist propagation requires a very fine mist or fog of high quality water, bottom heat, and a porous propagation media to provide the high humidity, warmth, and oxygen needed for leafy cuttings to root. Cuttings are taken from actively growing plants and work best with at least two nodes and two lateral (prompt) buds.

A balance must be achieved in tissue maturity. Although the mother vines should be actively growing, cuttings from the most succulent tissues often fail to root. Conversely, once the cuttings become too greatly lignified, the lateral buds have often abscised, and cuttings may fail while the dormant (latent) bud goes through a required dormancy before growing.¹³

In the field (UC Davis, CA), suitable tissue can be collected from most grape selections between early June and late August. Alternatively, mother vines can be maintained in a greenhouse and will provide excellent cuttings for propagation as long as there is sufficient light for active growth. We have had excellent success propagating from seedlings and newly rooted plants as long as these plants are kept growing actively with supplemental light and heat as needed.

There are many appropriate media for mist propagation including vermiculite, perlite, sand, rock wool, and cellulose sponges. These media must maintain moisture and provide oxygen root development. By itself, vermiculite, a micaceous mineral expanded with heat, can become too wet and compacted over time, leading to low oxygen levels and poor rooting. Perlite, a gray-white silicaceous material mined from lava flows, which is also expanded with heat, holds less water and provides a less "soggy" environment, but provides poor cation exchange leading to poor nutrient uptake of rooting cuttings. We have used both one-to-one mixes of vermiculite and perlite and pure perlite mixes with great success. Coarse sand can also be used, but the light-weight, pest-free nature of vermiculite and perlite make them superior.

Some grape varieties and rootstocks will produce roots (under mist propagation) but fail to establish after potting in greenhouse soil mixes or upon transfer to the field. Such failure is often due to root damage upon removal from the propagating substrate. In these cases, cellulose sponge or rock wool are better media, because they maintain strong root attachment to the cutting stem, resulting in greatly improved rooting. We have increased the establishment of new plants from green cuttings of hard-to-root Vitis species like V. cinerea and V. monticola by three to five fold with cellulose sponge cores (Grow-Tech Techniculture, San Juan Bautista, CA).

Rooting hormones do not dramatically improve rooting success of green cuttings from most V. vinifera varieties. However, they are very beneficial for rooting other Vitis species and some rootstocks. The auxins indolebutyric acid (IBA) and napthaleneacetic acid (NAA) are commonly used as five- to 10-second dips at 1000 to 3000 ppm.

Warmth, humidity, and light are critical for success with mist propagation. Temperatures in the 28º to 30ºC range are ideal. Humidities are kept constant and high using either electronic timers that discharge about five to 10 seconds of mist every five to 10 minutes or moisture-sensing devices, such as the "mist-a-matic" (Hummert Intl., Earth City, MO), that detect moisture levels below a set point and discharge the mist to compensate.

Moisture sensing devices can be difficult to set up but more than compensate for this effort by preventing moisture levels from getting too high during cloudy weather or evenings. Excessive water logging can be a serious limiting factor to success with mist propagation. As long as humidities are controlled, high light levels produce optimum rooting. Normal greenhouse shading levels with adjustments for climate are needed.

Green-grafting

Herbaceous grafting under greenhouse and in vitro conditions has been practiced with cucurbits and solanaceous plants for many years.¹⁴, ⁹ This process involves placing a shoot tip or meristem on the freshly cut surface of a seedling epicotyl, where it is held in place while it heals under high humidity conditions. Green-grafting has also been done in the field by grafting green scions or buds onto green shoots. This technique has worked with grape, but has not been widely utilized.⁵

Grapevines have been green grafted under greenhouse and in vitro conditions. Green-grafting has the advantage of being easy, having high success rates, and is capable of overcoming the graft incompatibility sometimes experienced between distantly related Vitis species.¹,¹⁸ This technique has been used to screen for virus resistance and more recently as a means of evaluating virus presence in certification programs.¹⁷,²⁰

Green-grafting has also been promoted as a method to rapidly produce grafted plants. In this process, greenhouse-grown mother vines of rootstocks and scions can supply the needed cuttings for year-round grafting. Plants can also be produced without being exposed to the field, greatly reducing the chance of virus and pest infection. Several California nurseries are producing plants with this process.

Success with green grafting is very dependent upon high-quality cuttings. We have obtained the best success with partially lignified cuttings from field-grown mother vines. It is possible to have good success with cuttings from greenhouse-grown mother vines, but only when conditions are optimized with high light, moderate humidity, proper fertilization and nutrient balance, and fungal and insect pest control. It is extremely difficult to produce high-quality cuttings during the winter months even with supplemental light and heat. In addition, greenhouse mother blocks are most successful when they are regularly repropagated into fresh support media.

The scion cuttings should have a full-sized leaf, which is trimmed by about half, and an active lateral (prompt) bud. The rootstock cuttings need to be at least 25 cm long, so that adequate spacing between the roots and graft union can be achieved for field planting, and should also have a trimmed leaf and an active lateral bud.

Most often the rootstock and scion cuttings are fitted together with a cleft graft which makes a slit down the center of either the rootstock or scion, while a long tapered wedge cut is made on the opposing piece. Razor blades or scalpels are ideal for making this graft, and the process has also been mechanized (La Haltogel Greffenvert, Mumm & Perrier-Jouët, Vignoble et Recherches, Épernay, France). Parafilm, perforated plastic bandage tape, or small clips can be used to hold the rootstock and scion in place while the union heals.

Once the graft has been made, the union must be healed under conditions of moderate light, high humidity, and good air circulation. The grafted plants can be placed in perlite, rock wool, or cellulose sponge. One technique recommends that the union be healed by placing the grafted plants into a small closed plastic tent with moderate temperatures and low light. Over several weeks, more light is provided and humidities are decreased. Fungal diseases thrive in this environment, and they must be controlled with fungicides. Alternatively, the grafted plants can be healed and rooted under the mist propagation conditions described above. In cases where rootstocks or species are more difficult to root, it is advisable to pre-root these cuttings under mist propagation prior to grafting.

Over a two- to four-week period, the union heals, the rootstock roots, and the plant is ready to establish for a month or more under greenhouse conditions. Following a "hardening-off" period, the green-grafted plants can be planted in the vineyard or continue growing in the greenhouse to become dormant before planting the following year.

Green-grafting produces an excellent graft union which appears more seamless than traditional bench-graft unions made with omega or dado saw cuts. However, after several years no apparent differences can be detected in health or nature of the graft union when compared with traditionally grafted plants. At the time of field planting, green-grafted plants normally have limited carbohydrate reserves and relatively weak root systems; they require careful care and watering for successful establishment.

Tissue culture propagation

Grapevines can be propagated fairly easily in tissue culture. Meristem or shoot tip tissue culture has been used to eliminate grape viruses in certification programs. Grapes have also been grafted under tissue-culture conditions to detect and evaluate resistance to viruses.18 Tissue culture has also been suggested as a means for rapidly propagating scarce or hard-to-propagate planting stock of clones or varieties.¹¹,¹⁰,⁸ The techniques are straight forward,¹² but they are practiced skills requiring substantial amounts of time and labor.

For routine elimination of viruses, shoot tips can be taken from actively growing plants in the greenhouse or field. As in the case of mist propagation, the high-quality of the plant material from which the tips are cut is essential to the success of the work.

Under sterile conditions, fresh cut tips are gently sterilized in a mild bleach solution with a few drops of detergent, rinsed, and then excised under a dissecting microscope.

The tips are placed on an initial media for six weeks, followed by rooting media. After about a month on rooting media, most shoot tips will be ready for transplanting. The roots are trimmed, and the young plants are transferred to sterile soil mix in a closed container. Air circulation is gradually increased after new growth occurs. After about one month, the plant is ready to be moved from the growth chamber to a greenhouse.

When plant material is severely limited or in high demand, additional meristems could be taken from these plants at this time. However, it would be more economical, in terms of time and money, to begin mist propagating herbaceous cuttings at this point. In most cases, it is more practical to begin mass propagation after disease testing has insured that virus has been eliminated.

ELISA and PCR testing can be used as the plants become established in the greenhouse. Woody indexing, still the definitive virus disease tests for the California registration and certification program, can begin during the first dormant season following tissue culture; normally the new tissue culture explants (TCEs) are at least six months old before they are woody indexed. We have found that initial screening of plants with laboratory tests allows us to eliminate some of the TCEs from the secondary testing process.

Green-grafting for virus indexing has proven useful to many labs for virus disease testing.²⁰ It has not proved practical in our hands. Although some tests can be run more quickly with green-grafting, the technique requires extremely high quality, year-round greenhouse space and is very labor-intensive.

Further, the green-grafting technique is not accepted by the California Department of Food & Agriculture or the U.S. Quarantine officials as a substitute for woody indexing, thus there are regulatory barriers to its use. It is probably most valuable in climates where the woody field index cannot be accomplished routinely. In Davis, the woody field index produces reliable results in two years, and winter cold is not a factor in completing the test.

We expect the most rapid progress in shoot tip culture is the elimination of viruses to come from developments in PCR detection of grapevine viruses, which should result in early, accurate screening of TCEs for disease. Ultimately, these PCR tests are likely to eliminate the need for either woody or green-graft indexing.

In vitro-grafting as a tool for propagation can be done in a similar manner to the green-grafting described above for virus.⁴ For difficult to root Vitis species and hybrids, it is possible to optimize rooting and cultivation treatments in vitro to an extent that is impossible on a mist bench. Some countries (Chile and South Africa, for example) that have established strict phytosanitary rules for grapevine importation have made it relatively easy for in vitro plants to pass through quarantine. By establishing a new selection in tissue culture, an importer might save months or even years of time that would be lost before traditional materials cleared quarantine.

Outlook for rapid propagation methods

Rapid propagation methods are very important when plant materials are limited due to the scarcity of a clone or variety or due to dramatic expansion in acreage. Much of the grape-growing world is currently experiencing both of these circumstances. New clones and varieties are in high demand, and there seems to be boundless demand for the fruit from new wine and table grape vineyards.

The most important aspect of the rapid propagation methods discussed above is their ability to quickly provide relatively large amounts of planting stock to grapegrowers. However, these techniques are rarely competitive with traditional bench-grafting methods. The production of green-growing bench-grafts from dormant cuttings requires about eight to 10 weeks from grafting to field planting. It is rapid, provides high- quality planting stock, and can be relatively inexpensive. However, this technique depends on adequate numbers of mature mother vines of the needed rootstocks and scions, and these require three to four years to reach maturity in terms of their wood production.

Rapid propagation methods allow growers to get new planting materials two years earlier than they would be available via traditional propagation methods. However, there are potential problems with these techniques. First, there are great differences in rapid propagation practices compared with traditional techniques, and therefore knowledge gaps could lead to uneven plant quality.

The second problem relates to growers and their ability to alter establishment practices to deal with a planting stock with lower carbohydrate reserves and with much less tolerance for uneven irrigation or rainfall. It is also important to time plantings of these rapid propagation materials so that there is sufficient time for roots to develop and lignify and for basal dormant (latent) buds to complete maturation, so winter cold damage is prevented.

There are other problems coincident with rapid expansion of vineyards. Limited planting stock and high demand have led to errors in labeling and distribution. The quality of planting stock can often be uneven, as demands on nurseries outstrip the normal limits to their production capacity. Growers are also less inclined to check materials and may be less likely to demand high quality when they are desperate to plant.

Rapid expansion in vineyard acreage also brings many new growers into the grape business, often without the knowledge or background to handle planting stock properly. Instructions from nurseries and viticulturists regarding storage and handling before planting, soils, proper hole size, irrigation schedules, fertilization, protection from vertebrate and insect pests, and training may not be understood or followed. Rapid vineyard expansion also brings great increases in diseases such as young vine decline, a complex of wood-rotting fungi (Phaeoacremonium spp.), that, when associated with weakened planting stock or poor vineyard establishment, can damage vines.¹⁵,¹⁶

Whether purchasing traditionally or rapidly propagated planting stock, the grower is well advised to make contact with highly regarded and trusted nurseries. Close associations with nurseries will result in better monitoring of planting stock and/or avoiding improper grower practices that result in poor vineyard establishment.

It is doubtful that rapid propagation techniques will supplant traditional grafting and propagation methods; however, these techniques can provide scarce materials in a more timely fashion and will greatly advance vine improvement and certification programs. Rapid propagation will always be more common during periods of burgeoning vineyard expansion. These materials can meet grower’s needs, but care must be exercised to meet the requirements of such planting stock.

Edited from presentation to the Tenth Australian Wine Industry Technical Conference, Sydney, Australia, 1998.

References:

1. Bouquet, A. and Hevin, M. 1978. "Green-grafting between Muscadine grape (Vitis rotundifolia Michx.) and bunch grapes (Euvitis spp.) as a tool for physiological an pathological investigations." Vitis 17: 134-138.
2. California Association of Winegrape Growers. 1998. "Grape acreages for 1997 increase." The Crush 26 (May Issue 6).
3. California Agricultural Statistics Service. "Grape Acreage Report." PO Box 1258, Sacramento, CA 95812.
4. Cantos, M., Ales, G. and Tronocoso, A. 1995. "Morphological and anatomical aspects of a cleft micrografting of grape explants in vitro. Acta Hort." 388:135-139.
5. Carlson, V. 1963. "How to green graft grapes." Calif. Agr. Ext. Publ. AXT 115.
6. Cirami, R.W. and Ewart, A.J.W. 1995. "Clonal selection, evaluation, and multiplication in Australia." pp. 52-59. In: Proc. Intl. Symp. Clonal Selection. J.M. Rantz Ed., Am. Soc. Enol. Vitic.
7. Golino, D.A., Sim, S.T., Grzegorczyk, W., and Rowhani, A. "Optimizing tissue culture protocols used for virus elimination in grapevines." ASEV: in press.
8. Gray, D.J. and Fisher, L.C. 1986. "In vitro propagation of grape species, hybrids and cultivars." Proc. Ann. Meet. Fla. State Hortic. Soc. 98:172-174.
9. Hartmann H.T., Kester, D.E., Davies Jr., F.T. and Geneve, R.L. 1997. Plant Propagation: Principles and Practices. Sixth Edition. Prentice Hall Inc.
10. Lee, N. and Wetzstein, H.Y. 1990. "In vitro propagation of muscadine grape by axillary shoot proliferation." J. Am. Soc. Hort. Sci. 115:324-329.
11. Lewandowski, V.T. 1991. "Rooting and acclimatization of micropropagated Vitis labrusca ‘Delaware.’" HortScience 26: 586-589.
12. Monette, P. L. 1988. "Grapevine (Vitis vinifera L.)." pp 3-37 In: Biotechnology in Agriculture and Forestry, Vol. 6, crops II. Ed. Y.P.S.Bajaj. Springer-Verlag.
13. Mullins, M.G., Bouquet, A. and Williams, L.E. Biology of the Grapevine. Cambridge University Press.
14. Oda, M., Nagaoka, T., Moi, T. and Sei, M. 1994. "Simultaneous grafting of young tomato plants using grafting plates." Scientia Hort. 58:259-264.
15. Scheck, H.J., Vasquez, S.J. Gubler, W.D. and Fogle, D. 1998a. "First report of three Phaeoacremonium spp. causing young grapevine decline in California." Plant Dis. 82: 590-590.
16. Scheck, H.J., Vasquez, S.J. Gubler, W.D. and Fogle, D. 1998b. "Young grapevine decline in California." Pract. Winery & Vineyard (May/June) 1998: 32-38.
17. Tanne, E., Shlamovitz, N., and Spiegel-Roy, P. 1993. "Rapidly diagnosing grapevine corky-bark by in vitro micrografting." HortScience 28: 667-668.
18. Walker, M. A. and Meredith, C.P. 1990. "The genetics of resistance to grapevine fanleaf virus (GFV) in Vitis vinifera." Vitis Special Issue 1990:228-238.
19. Walker, M.A. 1992. "Future directions for rootstock breeding." pp. 60-68. In. Proceedings of Rootstock Seminar: A Worldwide Perspective. Ed. J.A. Wolpert, M.A. Walker and E. Weber. Presented by the American Society for Enology & Viticulture, Reno NV June 24, 1992.
20. Walter, B., Bass, P., Legin, R., Martin, C., Vernoy, R., Collas, A. and Vesselle, G. 1990. "The use of a green-grafting technique for the detection of virus-like disease of the grapevine." J. Phyotopath. 128: 137- 145.