LIQUID POLLINATION IN PINUS

by

G.B. Sweet, R.L. Dickson, Yetty G. Budi Setiawati & Iskandar Z. Siregar
School of Forestry, University of Canterbury
Christchurch, New Zealand

INTRODUCTION

In a recent publication (Sweet et al, 1992), we reported on the potential of liquid pollination for making controlled crosses in Pinus radiata. In this article we review the rationale for using the technique, and describe in more detail how it is carried out.

One of the great virtues of liquid pollination in Pinus is that the application technique provides both the pollen and the pollination droplet at the same time. Unlike dry pollen application, pollen applied in a liquid suspension does not stay on the micropylar arms for several days waiting for a droplet, but is carried immediately to fill the micropyle. To us that offered a significant advantage, because we were seeking to carry out controlled pollinations without the use of isolation bags. We thus used compressed air to blow off existing (wild) pollen from the micropylar arms, and then applied the controlled pollen in water to fill the micropyle with the pollen which we wanted. By using dyed pollens we have shown that, if the application technique is good, all of the pollen in the micropyles can be that which has been applied. That is, “wild” pollens can be prevented from pollinating, without necessitating the use of isolation bags (Sweet et al, 1992).

We are collaborating with NZ Forest Research Institute to use techniques of genetic analysis to confirm that the system is effective on an operational scale. If it is, it will be valuable in making controlled pollinations in orchards, for two reasons. One, it improves enormously the economics and logistics of making controlled pollinations; but perhaps more importantly, by operating without isolation bags, the seed yields per cone are greatly increased. This second factor reflects the widely reported substantial negative impact of isolation bags on seed yields per cone.

HOW EFFECTIVE IS THE TECHNIQUE ?

Our data (see Table 1) come from pollinations without bags. Firstly, while liquid pollination achieved seed yields per cone which are comparable with those from normal (dry) pollination, it also achieved larger seeds with a higher germinative energy and capacity. What this means, we suspect, is that in Pinus, liquid pollination is biologically more effective than dry pollination.

In our view, the technique has a clear potential for use in controlled pollinated (CP) orchards; and the highly stocked “meadow” CP orchards developed in New Zealand are ideally suited to its use. Today, only a few seed orchards in the world are controlled pollinated, despite the considerable genetic advantages of the technique (see Sweet and Krugman, 1977). While this situation is probably in the process of change, it is pertinent to ask whether liquid pollination also has a place in open pollinated (OP) orchards? A number of OP orchards today are using supplemental mass pollination (SMP), with the intent of increasing the proportion of genetically improved pollen in the orchard (relative to wild pollen blowing in from outside the orchard). It would seem very logical for SMP applications to be made in liquid suspension, for the same reason that liquid pollination is effective in controlled pollinations made without isolation.

Table 1. Seed data for matched cones pollinated with liquid and dry pollens, respectively. Data are means of 10 cones, representing 3 different clones

Treatments with different letters differ statistically at the 1% level.

WHAT IS THE TECHNIQUE FOR APPLICATION OF POLLEN IN LIQUID ?

We use what is known as an air brush, or a mini-spray gun (see Figure 1.). Compressed air exiting from one jet passes over a second jet which is the outlet from the container with the pollen suspension. This process utilises the “Bernouli effect” to lift the pollen suspension and carry it onto the strobili. On a commercial scale, the compressed air source used can be a tank of the sort used for under-water diving, or a battery-powered compressor unit. If used with a conventional pressure regulator, the air pressure can be controlled to an appropriate level. A switch in the hand-piece allows air to be blown onto strobili with or without the inclusion of the liquid/pollen suspension.

Figure 1. Examples of spray guns of the type used for liquid pollination

HOW IMPORTANT IS THE APPLICATION TECHNOLOGY ?

As with any pollination, the pollen, irrespective of whether fresh or stored, needs to have a high germination percentage. In terms of concentration, our research shows that a suspension of 2.5 to 3.0 grams of pollen per 100 mls of water is needed for optimal pollination results. Lower concentrations than that can result in reduced seed yields. Water needs to be distilled or deionised, as pollen germination is frequently inhibited in domestically-supplied water. While the timing of application is not particularly important, several successive pollinations give better results than a single one. Data to support these statements is presented in Sweet et al (1992).

In New Zealand we frequently use pollen which has been dried (to between 6% to 12%) and stored for a year. Such pollen needs to be re-hydrated before mixing it into water. We do this by incubating it in a humid atmosphere for 4 – 12 hours.

ARE THERE FUTURE DEVELOPMENTS WITH THE TECHNIQUE ?

Obviously one of the first questions we asked was whether water was the optimal liquid in which to suspend the pollen ? (The pollination droplet itself is a solution of simple sugars - McWillliam, 1958). We have done a lot of laboratory testing to determine whether by using solutions other than pure water, we can improve the in vitro germination of pollen. We have managed to improve germination by more than 15% and pollen tube growth in vitro by some 20%, by the use of appropriate mixtures of hormones, mineral ions, carbohydrates, etc. We have experiments in place to determine whether these additives, when used in vivo, will translate into improved seed yields per cone; but the results are not yet available. A recent perception in New Zealand is that infection of strobili with pathogenic bacterial and fungal spores may take place at the time of pollination; and again, we have tests in place to examine seed yields following the application of pollen in suspensions of antibiotics and fungicides.

We have not attempted pollination with species other than radiata pine, but we see no reason why it should not be successful. In Pinus species the pollination droplet allows pollen grains to float upwards, against the force of gravity, into the micropylar cavity. There is some evidence (Greenwood, 1986) to suggest that in nature this flotation may often occur in water from raindrops, rather than in the pollination droplet which appears to occur only infrequently, and under very humid conditions. Thus there is nothing biologically unnatural in applying water immediately after pollen dispersal. We have in fact tested that technique, but found that application in water is more effective than applying the pollen dry, and applying the water subsequently.

Back in 1962, Allen and Sziklai reported a trial with liquid pollination on Douglas fir. Because Douglas fir pollen does not have wings, and there is no pollen droplet mechanism (Allen and Owens, 1971), the biology of pollination is very different from that in Pinus. But nonetheless, the authors reported numbers of filled seeds per cone which were comparable to or better than those from dry pollination. Again, it may be suggested that rainfall before or after pollination may be a common natural event, and that liquid pollination may somewhat mimic this in a number of species.

The commercial application of liquid pollination is still in its early days, but there are encouraging signs to suggest that its use may become widespread. Already, interest has been expressed in using the technique for hybrid pollination in Pinus.

References

Allen, G.S. & Owens, J.N. (1972). The life history of Douglas fir. Environment Canada, Ottawa.

Allen, G.S. & Sziklai, O. (1962). Pollination of Douglas fir with water suspensions of pollen. Forest Science 8, 64–65.

Greenwood, M.S. (1986). Gene exchange in loblolly pine: the relation between pollination mechanism, female receptivity and pollen availability. Amer. J. Bot. 73(10) 1443–1451.

McWilliam, J.R. (1958). The role of the micropyle in the pollination of Pinus. Botanical Gazette 120, 109–117.

Sweet, G.B. & Krugman, S.L. (1977). Flowering and seed production problems - and a new concept of seed orchards. Invited Special Paper, Proceedings 3rd World Consultation on Forest Tree Breeding.

Sweet, G.B., Dickson, R.L., Donaldson, B.D. & Litchwark, H. (1992). Controlled pollination without isolation - a new approach to the management of radiata pine seed orchards. Silvae Genetica 41(2), 95–99.

Forest Genetic Resources Information no. 21. FAO, Rome (1993).
Manuscript received June 1993