Indra S. Harry

Somatic Embryogenesis and Plant Regeneration from Mature Zygotic Embryos of Red Spruce

Somatic embryogenesis and plantlet regeneration were obtained from mature embryos of red spruce, Picea rubens Sarg. Embryogenic tissues were produced from both 5- and 20-yr-old seed collections. The basal medium used was von Arnold and Erikssons (AE) salt formulation supplemented with 1,000 mg/L casein hydrolysate, 250 mg/L L-glutamine, 10 μM each of naphthalene acetic acid and N6-benzyladenine (BA), 2% sucrose, and 0.3% Gelrite Gellan gum. Tissues were incubated in the dark at 26 C. Longterm cultures were maintained on the above medium, but with 5 μM BA. Tissues were cultured on phytohormone-free basal medium containing 1% activated charcoal for 7 d, and for maturation were transferred to basal medium containing 20-50 μM abscisic acid. When embryos appeared to be morphologically similar to fully imbibed zygotic embryos, they were removed for germination. They were partially dried and germinated on 1/2 AE at 24 C under reduced light. Plantlets were transferred to sterile peat moistened with 1/4 AE for further growth.

Optimization of bud induction in cotyledonary explants of Pinus canariensis

A method is described for using liquid pulsing as an alternative to the conventional induction protocol for Pinus canariensis. Using Day 0 and Day 3 explants, the best exposure time was 8 h and 4 h respectively, in a non-buffered 100 μM N6-benzyladenine solution, followed by culture on half-strength Bornmans medium containing 3% sucrose and 0.8% Difco BactoR agar. With this procedure, 97% of the cotyledonary explants produced about 14 buds/explant. These results were comparable to a 14-day induction period on full-strength Bornmans medium containing 10 μM N6-benzyladenine.

In vitro culture of forest trees.

Forest resources are disappearing at an unprecedented rate. An important component of forest ecosystems are trees, which provide food, fuel, construction and industrial products. In addition, trees are recognized as the critical elements in maintaining stability in the world’s atmosphere. Currently, a major concern is the erosion of genetic variability for a given species. Therefore, both in situ and ex situ management is necessary for biodiversity, conservation and consequently for tree crop improvement (NRC, 1991).

Plantlet Regeneration from Cultured Embryos and Seedling Parts of Red Spruce (Picea rubens Sarg.)

Plantlet regeneration of red spruce (Picea rubens Sarg.) was achieved from mature embryos, cotyledons, and hypocotyl explants. Optimal bud induction was observed on half-strength modified Bornmans (MCM) medium with 10 μM N6-benzyladenine for 14 d. Shoot elongation occurred after transfer to half-strength modified von Arnold and Erikssons medium. Decreasing sucrose to 2% and addition of conifer-derived activated charcoal enhanced shoot elongation. Low concentrations of cytokinin (0.1 μM) promoted growth of axillary shoots. Rooting was induced on excised shoots by dipping in rooting powder or pulsing in high concentrations of indolebutyric acid plus indoleacetic acid or naphthaleneacetic acid prior to transfer to hormone-free medium. Plantlets grew after transfer to the greenhouse

Plantlet regeneration in chir pine (Pinus roxburghii Sarg.): morphogenesis and histology

Abstract In the first of this two part study, various factors affecting morphogenesis including media, age of explant, phytohormones and environmental factors were tested. Half-strength Bornmans medium (MCM) was superior to all the other media tested. Although N6-benzyladenine at 10 μM was the best for bud induction, bud development was enhanced when cotyledons were cultured on this treatment for 7 days, and then transferred onto medium containing 10 μM zeatin for an additional 7 days. Half-strength Schenk and Hildebrandt medium produced the highest shoot elongation index and was used from the sixth week to about the fifteenth week of culture. Shoots excised after this time elongated faster on half-strength MCM solidified with Gelrite®. On average, four axillary shoots were obtained by decapitation of a 28-week-old adventitious shoot. Eighty-one percent rooting was obtained by incubating 28-week-old shoots in 1 4 MCM containing 0.5 μM napthalene acetic acid for 4 weeks. In the second part of the study, the anatomy of bud formation and the histology of the root-shoot junction were examined. The anatomical events culminating in shoot formation in 30 days included anticlinal and periclinal mitotic figures, meristemoid formation, meristematic domes and juvenile leaf primordia. The plantlets had a solid root-shoot junction, and 85% survival was obtained after transfer to the greenhouse.

Special Problems and Prospects in the Propagation of Woody Species

Woody plants represent a vast array of types relative to their taxonomy or use and include both angiosperms and gymnosperms. In general, these are more difficult to propagate asexually than herbaceous species, which in part is related to the phase change from juvenility to maturation that most of them undergo. Thus, with few exceptions, methods for the large-scale regeneration of true-to-type clones are limited. This is particularly true for forest trees, the woody plants discussed in this article.

Regeneration of plantlets through organogenesis from mature embryos of jack pine

A protocol is described for the production of plantlets from mature excised embryos of jack pine (Pinus banksiana Lamb.), a conifer widely distributed in temperate North America. Shoot buds were induced on von Arnold and Ericksons or Bornmans MCM salts with 10 μM cytokinin for 2 weeks, using Phytagar® for gelling the medium. Bud development and shoot elongation required frequent subculture on MCM medium with activated charcoal and reduced inorganic nitrogen during elongation. Shoots were rooted in peat-perlite with α-naphthaleneacetic acid. The protocol produces about six plantlets per embryo.

Organogenesis and somatic embryogenesis in Cupressus sempervirens

Adventitious buds of Cupressus sempervirens L., were formed on excised mature embryos cultured for 10 days on half-strength Quoirin and Lepoivre medium (1/2QP) with 10 μM N6-benzyladenine. For shoot development, embryos were transferred to 1/2QP without growth regulators. Axillary shoot formation and rooting occurred spontaneously as adventitious shoots aged and transfer intervals were increased. Embryogenic tissue was obtained from immature embryos on induction media consisting of von Arnold and Eriksson (AE) or Gupta and Durzan (DCR) salts with 10 or 20 μM 2,4-dichlorophenoxyacetic acid. Cultures were maintained on DCR with 5 μM α-naphthaleneacetic acid and 5 μM BA.

Effect of various bud induction treatments on elongation and rooting of adventitious shoots of Canary Island pine (Pinus canariensis)

Three-day-old cotyledonary explants of Pinus canariensis were subjected to 30 induction treatments using half-strength Bornmans medium containing various combinations of N6- benzyladenine, zeatin, kinetin and 2-isopentenyl-adenine. The highest numbers of buds were obtained with 10 μM 6-benzyladenine, but both kinetin and zeatin influenced shoot elongation. Shoots were maintained on half-strength Schenk and Hildebrandt medium with 2% sucrose and 0.05% activated charcoal. For rooting, shoots were pulsed for 4 h in a 100 μM indole-3-butyric acid aqueous solution (pH 4.2–4.5), and planted in peat:vermiculite:perlite (1:1:1). After 8 weeks, the numbers of rooted shoots were similar for most treatments. Therefore, the bud induction treatments did not significantly influence rooting of adventitious shoots of Canary Island pine.

Regeneration of plantlets from mature embryos of western larch

SummaryLarix occidentalis Nutt. can be micropropagated using whole excised mature embryos. The basal medium for induction (over 70% response) was half-strength Quoirin and LePoivre salts with Schenk and Hildebrandt organics and N6-benzyladenine singly or with kinetin at a final concentration of 10µM. Bud elongation was best on half-strength Schenk and Hildebrandt or Bornman’s mineral salts, and elongated shoots could be maintained on either half-strength Quoirin and LePoivre or Schenk and Hildebrandt formulations containing 0.05% activated charcoal, 2% sucrose, and 0.7% agar. Axillary buds developed naturally on elongating juvenile shoots, but a high sucrose treatment (6%) for 2 wk enhanced the number produced. Very good rooting (80 to 100%) was obtained by pulsing shoots for 2 to 3 h in a solution of indole butyric acid (1 mM, pH 4.5–5.0), or by planting shoots in peat-vermiculite moistened with basal medium containing 5µM α-naphthalene acetic acid, pH 5.0. Rooted shoots were hardened before transfer to the greenhouse, and initially were kept at low light and high humidity after transfer to ex vitro conditions.