Marlborough at 50: a new project to safeguard the vineyard of the future, using accelerated clonal selection

So the Marlborough wine region turns 50 this year. With its continued growth and success, you could forgive the region for resting on its laurels. But this isn’t that attitude encountered when you speak to people based in the region. New Zealand’s wine industry isn’t taking anything for granted, and is well aware how highly exposed it is with just one grape variety driving so much of the export trade, which it relies heavily upon.

The success of New Zealand as a wine country has largely been down to its ability to produce a unique style of Sauvignon Blanc that is highly in demand globally. Since the late 1980s when this style emerged on an international stage, the demand hasn’t slowed, and there’s no reason to suspect it will. Still, it is wise to plan now to prevent any unpleasant surprises in the future.

There are four potential threats to Marlborough as a wine region, and by extension New Zealand as a wine-growing country. First, the world might fall out of love with Sauvignon Blanc. Second, other countries might be able to produce Sauvignons in a Marlborough style at a lower price. Third, that brand ‘New Zealand’ is damaged by low quality wines made by cowboy operators swooping up above-yield-cap/above-disease-threshold fruit at bargain prices and then selling the wine it makes merely on the basis of price. Fourth, that climate change might render Sauvignon Blanc less well adapted to the conditions in Marlborough.

Concerning the first three threats, there is little that can be done. But the fourth – designing the Marlborough vineyard of the future – is definitely something that can be worked on. But unlike other crops, where breeding programs can be used to produce new plants, when it comes to grape varieties these are lost when breeding takes place. Breeding involves sex, and the reason sex is so prevalent in nature (when cloning or vegetative reproduction would be so much easier and more efficient) is because it is a brilliant way of introducing variation. Consider your parents: what they look like, their physical and mental characteristics – and then look at you, and if you have siblings, them too. There are some similarities, for sure, but there are lots of differences. Sex has introduced variation, and in nature this is vital for two key reasons: disease and a changing environment. To use rather loose scientific speech, evolution needs organisms to keep changing, to provide the raw material that can drive differential reproductive success, the ability to adapt to changing conditions, and also to keep one step ahead of disease organisms and parasites. In the absence of breeding, where does this diversity that allows for adaptation come from? This is the problem faced with grape varieties, which much be reproduced vegetatively, without sex.

Genetic variation without sex

Help is at hand in the form of a genetic feature that’s both hard to explain, but also has played an important role in the evolution of plants. This feature? Transposable elements (TEs), also known as jumping genes. These were first discovered in maize in 1950 by a researcher called Barbara McClintock: it turns out maize has a particularly TE-rich genome, one that has been described as islands of genes surround by a sea of nested TEs. She referred to these TEs as ‘controlling elements’ because of the way they can affect gene expression. McClintock showed that some of the genes producing pigmentation in maize seemed to be hopping round the genome: they weren’t consistently located on the same chromosome. Her research was initially treated with scepticism: the idea of jumping genes seemed like science fiction. But this ground-breaking work was rewarded with the Nobel Prize in 1983.

Transposable elements are sequences of DNA that can change their position in the genome. They move through the genome using a transposase enzyme (DNA transposons) or an RNA intermediate (retrotransposons; the DNA is copied to RNA and then back to DNA), and they have been important in shaping genome evolution. They make up 14% of the genome in Arabidopsis thaliana (the plant most widely used as a model organism in plant research) and 80% in maize. They have been described as engines of plant genome evolution. Transposable elements are natural molecular tools that can re-shape the genome, leading to rapid adaptation to environmental changes, and this is why they are of such interest to vine breeders who can’t use sex to introduce genetic variation.

Many of these TEs have accumulated stress response regulatory elements that switches them on in response to environmental stress, giving the plant a way of increasing variation when it’s most needed. The idea is that if all is well, then no change is warranted, and the plant can carry on doing what it’s doing. But when things change in the environment and put the plant in stress, some changes are potentially helpful. Of course, these changes might not be helpful, but the plant makes a gamble because doing nothing and carrying on as before is setting it up for failure. Doing something is better than doing nothing.

So, back to Marlborough. This is a region that has enjoyed a lot of commercial success with Sauvignon Blanc, and of the 30 000 hectares of vineyards in the region, some 80% of these are planted with Sauvignon. Sauvignon is the grape that put the country of New Zealand on the world map. And this potential problem is compounded by the fact that most of this is a single clone of Sauvignon Blanc, known as UCD1. While there is no suggestion that this single-varietal, single-clone focus is causing difficulties now, it restricts the region’s ability to adapt to any change in the future. If you want to keep a grape variety, but would like to find vine material better suited to site, or to a changing climate, then clonal selection is the only option (assuming that genetic modification is out of the question). Naturally occurring genetic diversity is the raw material of this clonal selection, but it can be a slow process finding new clones, and then testing them.

For this reason, a research project titled Sauvignon Blanc 2.0 was initiated 2022 at the Bragato Research Institute in Blenheim, that has NZ$18.7 m funding committed to it from Ministry for Primary Industries (MPI), New Zealand Winegrowers and a group of wine industry members. This aims to generate new genetic material for Sauvignon Blanc by making use of the vine’s ability to adapt in response to stress, and then to select the more desirable elements of this genetic diversity to produce new clones of Sauvignon that will give growers tools to create vineyards adapted to deal with future challenges, including climate change. These new clones might have increased productivity, show better disease resistance and have increased water use efficiency. Effectively, the project aims to use science to accelerate the speed of clonal selection and then filter out the best results.

The goal of this seven-year project is to create 12 000 new Sauvignon Blanc ‘clones’ and then screen them using a high-throughput third-generation DNA sequencer: the first one in the country. To make this screening possible genetic markers for desirable traits are needed, and so part of the project is focusing on identifying these. This screening vastly accelerates the whole process, and then the most promising clones that get through this screening can be trialled in the field.

The rationale behind this applied research project needs some explaining. It is based on the use of specific stress treatments applied to cultured vine tissue to increase the rate of genetic and epigenetic changes. The focus is on TEs and their regulation. These TEs become active in plant cells in response to environmental shock, and serve as a way for the plant to adapt to environmental changes. It is becoming clear from current research that most variation in grapevine clones is due to these TEs.

As an example, one study from 2012 by Grégory Carrier and colleagues looked at the differences between clones of Pinot Noir in France. They sequenced PN386, PN583 and PN777, and compared this with the sequence of PN115, using the reference PN40024 (the first grapevine genome sequenced, in 2007) to align all the sequences. Most of the variations were from insertion polymorphisms from mobile genetic elements. Similar studies have been done with Chardonnay, Zinfandel and Malbec, and one is currently underway looking at clonal variation in Chenin Blanc.

How to accelerate clonal selection in vines

The first stage of the project to speed up clonal selection is to carry out a process called somatic embryogenesis. The most common way to do this is to take a flower cluster, sterilize it by using a solution of a bleach (hypochlorite) plus a wetting agent (the detergent Tween) and then rinse with distilled water. The next stage is to remove the anthers, which are the structures from the grape flower that bear the stamens (the male sex organs). The excised anthers are then sterile-cultured on a medium containing nutrients, some sugar, and some plant hormones (an auxin and a cytokinin). This creates the growth of a mass of cells that are de-differentiated (they were cells of a certain type, but they have lost this differentiated state) and which are totipotent (that is, they can become any cell type). This ability to de-differentiate cells makes plants quite different from us: in mammals, the only cells that are totipotent are germ cells, which we have in our germline as we develop and which produce gametes. This separation of body (somatic) cells and germ cells doesn’t hold in plants. With further tweaks to the hormones in the growth medium, the dedifferentiated cells can then form embryos, and from these a grapevine can be grown. Anthers are most commonly used to initiate these cultures, but embrogenic callus can also be regenerated from stigmas, ovaries, sytles, whole flowers, leaf discs, tendrils or stem nodal explants.

The process of somatic embryogenesis itself causes reorganization of the TEs and their epigenetic modifications. This rearrangement is further accelerated if certain stresses are applied to the cells while they are in their undifferentiated state. In the New Zealand project, one of the stresses they tried is ultraviolet light. But they found that a much more effective way of creating genetic variation was to culture the cells with various species of yeast that were initially isolated from vineyards in New Zealand. The leader of the Bragato project is Dr Darrel Lizzamore, and in his PhD thesis from 2013 he discovered that co-cultivating these embryonic cells with live yeasts up-regulated the TE activity considerably. The idea here is that culturing somatic cells like this undoes all the programming of the genome, then adding a stressor means that reprogramming takes place during embryogenesis creating variation that may or may not be interesting.

To take these embryos and produce vines with them, and then let the vines grow for long enough to produce grapes would be an extremely lengthy process, taking several years. This would slow up this project and would add to its expense. This is where next generation DNA sequencing comes in. Genetic markers for desirable features in these new clones are vital for this work, because they speed it up massively. Then, the baby vines that pass this first screen will go on to the next stage, which is growing them in a vineyard setting to see whether the genetic changes are stable, and result in the desired output. This is where the project becomes more long-term in nature. If the new clone survives this second selection, there’s the likelihood that it would be given to nurseries to grow up and make available to winegrowers in the region. This is a very promising strategy for producing new clones of Sauvignon that will be better adapted to the vineyard of the future than the existing single clone. Importantly, this strategy doesn’t involve genetic modification, which is currently unacceptable in the eyes of many consumers.

But aside from this accelerated selection, there may already exist quite a bit of variation in the vineyards that have been planted for a while. This natural variation is the basis of existing clonal selection, but a lot of the variants – potential new clones – will be hidden away and won’t have been spotted by winegrowers. If there was a way to take these genetic markers and screen existing vine material, some surprises might emerge, especially in older vineyard where there have been lots of opportunity for modification.

Many of Marlborough’s existing vineyards are an approaching an age where replanting will soon take place. It would be nice to think that before too long the region will have extensive plantings of unique New-Zealand-developed clones of Sauvignon Blanc that will enable this variety to thrive, even in a drier, warmer and less consistent climate.