Novel Methods in Micropropagation

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Proc. 1st International Symposium on Accl. & Estab. Micropropagated Plants
Eds.: A.S. Economou & P.E. Read. Acta Hort. 616, ISHS 2003. p.71-76

Novel Methods in Micropropagation

John E. Preece Paul E. Read
Dept. of Plant, Soil & General Agriculture Dept. of Agronomy and Horticulture
Southern llIinois University University of Nebraska
Carbondale IL 62901-4415, USA Lincoln NE 68583-0724, USA
jpreece@siu.edu pread@unl.edu

Keywords: Bud encapsulation, juvenility, shoot forcing

Abstract
The focus of this paper is on two novel methods for micropropagation, forcing softwood shoots from large stem sections and encapsulation of buds and shoot tips for storage and facilitation of Stage IV, acclimatization. Neither method is being used widely by commercial micropropagation laboratories. Branches are excised from within the cone of juvenility of woody plants that were originally propagated from seed. The branches are cut into sections 30-50 cm long and placed horizontally in flats or bencbes filled with perlite. Irrigation must be done with care to avoid water contacting the stem segments or new softwood shoots. The softwood shoots are excised, surface disinfested and used as explants. If the softwood shoots are to be used as macrocuttings, forcing is best under intermittent mist. However, forcing in mist will result in microbial contamination if micropropagation is the goal. Alginate gel is used to encapsulate somatic embryos to produce synthetic seeds. The same gel matrix can be used to encapsulate buds and shoot tips from Stage II proliferating cultures. The encapsulated buds can be stored under refrigerated conditions or dehydrated and placed in liquid nitrogen. Additionally, shoots and roots can grow from the encapsulated shoot tips and buds and these can be transplanted to a greenhouse medium for acclimatization. Because of the nutrient salts and sucrose in the gel matrix, the incorporation of fungicides into the gel has reduced fungal growth during Stage IV and has increased the percent survival.

Introduction
Plants have been micropropagated commercially for decades. Many of the laboratories that are currently producing plants for production are successful and have developed their protocols based on past research. However, there are some techniques that are currently being developed that have the potential to impact commercial micropropagation in a positive manner. Breakthroughs are largely in the area of woody plant micropropagation, but may also be utilized in the micropropagation of herbaceous species.

The focus of this paper is on two of these newer techniques: forcing softwood shoots from large stem segments and encapsulation. Neither technique is in widespread use in commercial micropropagation laboratories, but both appear to have great potential and may become adopted by labs in the near future.

Juvenility
Plants in the juvenile phase of growth generally are easier to propagate vegetatively than plants that have reached the adult phase (Preece, 2002). Cuttings taken from adult shoots of plants can be rooted, but the frequency of success is low, especially with woody plants. Likewise, researchers have had great challenges when attempting to micropropagate adult forms of many woody species. In fact, the change from the juvenile to adult phase is considered to be the most serious constraint to rooting cuttings (Howard, 1990) and micropropagation of shrubs and trees. Most of the difficulty experienced in rooting mature shoots seems to be caused by their altered physiology, but can also be related to greater contamination with microorganisms and viruses than with juvenile forms.

Juvenile explants are usually most readily established in vitro, grow and proliferate at a more rapid rate, and are more rootable than adult material. This is especially true with tree species where mlcropropagatlon of adult material is often difficult. Explants from mature shoots more frequently produce dark, phenolic exudates in vitro and often suffer necrosIs than juvenile-source explants (Hanus and Rohr, 1987). Although seedling shoot and embryonic axis explants grew well on the same medium formulation, we observed shoot tip explant death within a few hours for adult Juglans nigra (eastern black walnut) (Preece and Van Sambeek, unpublished). It was only by changing the medium and culture conditions that we have been able to maintain adult J. nigra shoot cultures for years (Pearson and Preece, unpublished). We still cannot root adult origin microshoots of black walnut, although juvenile origin microshoots of black walnut can be rooted (Heile-Sudholt et al., 1986).

When breeding and selecting new, superior plants for clonal propagation, it is usually necessary to wait until the plant reaches maturity for full evaluation of the phenotype. This allows for assessment of important features, such as ultimate form and size, flowering and fruiting characteristics, autumnal coloration, and other traits. At the point that the mature phenotype is known, the plant is an adult and often becomes difficult to propagate clonally.

Cone of Juvenility   The majority of juvenile growth occurs when a plant is young and still exhibiting juvenile characteristics. As a plant ages, the older and lower parts retain their juvenile traits and the adult phenotype appears on the newest growth. On a tree, the trunk and lower branches that formed when the tree was young will retain their juvenile characteristics. Once the tree is older, the newest branches that form exhibit adult traits. Some temperate tree species, such as Quercus (oak), Fagus (beech), and some Acer (maple) retain their leaves throughout the winter as a juvenile characteristic. Viewing mature specimens of these species during the winter and focusing on the portion still holding its leaves, allows visua1ization of the shape of the tree when it was young and juvenile. This older, but more juvenile portion of the plant is called the “cone of juvenility”. A challenge is to be able to collect explants from within the cone of juvenilty. If water sprouts have formed from the lower stem, or suckers have grown from the roots on a large woody plant grown outdoors, they are frequently difficult or impossible to surface disinfest, but they can be harvested during the dormant season and forced in solution indoors. When such juvenile shoots are not available, people have even gone to the extreme measure of cutting down a tree to encourage juvenile shoots to grow from the stump or roots. When this is done, propagules often respond as well as juvenile cuttings
or explants.

Shoot Forcing
When practical, it is best to grow stock plants in a greenhouse or other controlled environment where they are isolated from the wind and rain. When outdoors, plants have microflora growing on them that are difficult to remove using surface disinfestation techniques. These microbes are often in tiny cracks and crevasses and the disinfestant may not penetrate these areas. A problem is that many woody specics are either too large or are growing in the ground and cannot be moved easily to a greenhouse or growth room. Therefore, it is often necessary lo remove parts of the plants such as branch tips or segments, usually during the dormant season, and force new shoot growth on them indoors or in a greenhouse environment. This new growth can then be excised, successfully surface disinfested and used for explant material. Although species specific,
these explants tend to be relatively contaminant-free, they may also be more readily established in culture than explants taken during spring and summer, and can be less prone to produce polyphenolic exudates.

We have been conducting research on forcing shoots for years. Much of the focus has been on shoot tips harvested from trees and shrubs during the dormant season. Commonly, 20-25 cm long stems (Read and Yang, 1991) are harvested from deciduous trees and shrubs and then soaked for 15 min in a 0.78% NaCIO solution with Tween 20 (Read and Yang, 1988, 1991; Yang and Read, 1992, 1993). Following the bleach treatment, a fresh cut is made at the base. The lower, cut ends of the stems are placed in containers with water, 200 mg 1-2 8-hydroxyquinoline citrate (8-HQC) (Read and Yang, 1987), sucrose and sometimes plant growth regulators. Yang and Read (1992) reported that faster bud break and more bud and shoot elongation was promoted if the stems received the 15 min soak in bleach solution prior to forcing compared lo those that did not.

A related technique is forcing terminal shoots in a greenhouse medium under greenhouse or other controlled environment. Onay (2000) harvested 3-4 cm long terminal lignified stem sections from 30 year-old pistachio trees and immersed the cut ends in plant growth regulator solutions, bcfore placing them in a greenhouse medium. New, softwood shoots were forced in the greenhouse under a 24 hour photoperiod until they were sufficiently large to excise, surface disinfest and place in vitro. Because these are excised from the tips of branches, the most adult portion of a plant, they can prove to be somewhat difficult to propagate vegetatively. A solution to this can be the use of plant growth regulators applied to the cuttings before placement in a greenhouse medium or by incorporation into the forcing solution. For example. in vitro shoot proliferation from shoot tips of Philadelphus and Dirca was increased by introducing BA into the forcing solution (Read and Yang, 1987). Yang and Read (1993) also found that more softwood explants from forced ‘Vanhoutte’s’ spirea stems produced shoots or more shoots were produced per explant if the forcing solution contained 44.4 uM compared to 4.4 uM BA. However, as GA3 concentration in the forcing solution increased from 2.9 to 145 uM, the number of explants that produced shoots decreased. When GA3 was in the forcing solution of American chestnut or Aesculus, there was no effect on in vitro performance of shoot explants. Therefore, the effects of plant growth regulators in the forcing solution are at least somewhat species and plant growth regulator specific.