An Improved Method for Fractionation of Small Quantities of Lettuce Latex

While large quantities of latex can be handled either by standard extraction techniques such as Soxhlet extraction or accelerated solvent extraction (ASE), smaller samples on the order of 0.3 - 0.5 g require handling on a microscale. We collected latex from lettuce plants in microcentrifuge tubes and, after drying under vacuum, resuspended the dried sample in acetone by holding in an ultrasonic cleaner. The resulting fine suspension was readily extracted with acetone and toluene to provide fractions representing the resin and rubber content of the latex. Using this approach, we compared latex from stems of bolting lettuce and from the floral stem of lettuce plants. While both types of stems contained a similar percentage of resin, the rubber content of the bolting stems exceeded that of the floral stems.


Introduction
In efforts to develop new sources of natural rubber, a number of plants have been evaluated. Many plants secrete a latex that contains natural rubber, but only a limited number produce a rubber of sufficient quality to be useful for producing rubber products such as tires [1]. In addition to the standard commodity rubber derived from Hevea brasiliensis Mull. Arg., Parthenium argentatum A. gray (guayule), Taraxacum kok-saghyz (Russian dandelion), Lactuca serriola L. (wild lettuce) and Lactuca sativa L. (salad lettuce) all produce a suitable rubber for commercial use, with the latter four species suited for growth in temperate climates [2] [3].
The rubber content of lettuce latices has been previously reported. The rubber content of wild lettuce ranges from 2% to 8% [4] while carefully washed latex particles from both wild lettuce and salad lettuce (cv. Salinas) contain 54% and 47% rubber content based on products from acetone and dichloromethane extraction [3].
As part of an effort to understand the biosynthesis and regulation of rubber production, we have initiated research on lettuce as a simple model system for rubber biosynthesis, as it has two month growth time to bolting, with the stem being a good source of latex. As such, we have investigated latex production in bolting and flowering lettuce.

Materials and Methods
Plant growth: Romaine lettuce seeds, Parris Island Cos [5], were obtained locally and sourced from Cornucopia Seeds (Felton, CA, USA). Seeds were germinated on wet soil (Sunshine Mix #1, Crop Production Services, Sacramento, CA, USA) with a light covering of sand, conditions that provided an 85% germination rate. When true leaves appeared and expanded, seedlings were transplanted to 18 liter pots containing the same soil with 50 ml of slow release fertilizer added (Osmocote, 15-9-12, 8-9 month, Scotts-Sierra Horticultural Products Company, Marysville, OH, USA, full composition provided at https://icl-sf.com/us-en/products/ornamental_horticulture/osmocote-plus-stand ard-8-9-a903266-15-9-12/). Plants were grown under our standard greenhouse conditions with a temperature range of 17˚C to 27˚C with ambient springtime light. Humidity was not controlled but generally ranges from 40% to 70%. The plants were maintained through bolting, flowering and seed maturity.

Results and Discussion
Previous authors have noted some difficulty with extraction of lettuce latex and possibly latex from other plants as well. Fox [6] noted the resin forms a brittle material and an extraction procedure described for wild lettuce [4] uses stirring with a spatula and an extended extraction time to extract rubber from the pellet.
The use of the ultrasonic cleaner pulverizes the hardened dry latex into fine particles and serves the same purpose as extended vortexing to extract the sample.
While Soxhlet extraction and advanced solvent extraction (ASE) are very effec- The results of the lettuce leaf stem vs. floral stem latex comparisons are presented in Figures 1(a)-(c), presented as the mean of 4 determinations for each latex type. The water content of each sample is close, with the means for each near 70% (Figure 1(a)). On the other hand, the floral stem has more resin, nearly 12% vs. 10% for the leaf stem (Figure 1(b)). The rubber content of the floral stem is lower than the leaf stem with a mean of 2.5% vs. 4.3% for the leaf stem (Figure 1(c)).
Rubber contents from each sample vary, with the range for the floral stem latex 1.3% to 3.7% rubber, while for the leaf stem the range is 2.8% to 6.7%. If the water content is removed from the equation, as in Bushman et al. [3], the rubber content of the highest rubber content leaf stem latex is 40% while the highest rubber content floral stem latex is 25%. In our experience, it is easier to collect latex from the leaf stem, and this appears to be the best source of rubber from lettuce. However, based on cross sectional area, the floral stem may have a higher percentage of laticifers, providing an advantage in biochemical studies.