Plant Cell Reports (1987) 6 :5-7 Plant Cell Reports © Springer-Verlag 1987

Plant regeneration from eotyledon-hypocotyl explants of Pinus strobus L.

Karan Kaul

KSUCRS Plant and Soil Science Research, Kentucky State University, Frankfort, KY 40601, USA

Received October 13, 1986 / Revised version received December 2, 1986 - Communicated by I. K.Vasil

ABSTRACT

Rooted adventit ious shoots were obtained from cotyledon-hypocotyl explants of asept ica l ly grown seedlings of Pinus strobus L. The explants consisted of the top ~ 3 mm of the hypocotyl attached to the whorl of cotyledons which had been trimmed to about hal f of the i r or ig inal length. The explants were cultured on a modified Murashige and Skoog's medium containing 0.2 mg/L NAA and 2 mg/L BA for 2-3 weeks and then on the medium of Li tvay et a l . without any growth hormones for 8 to i0 weeks to obtain shoot induction and shoot growth. Root induction and root growth were achieved by cul tur ing the shoots on hal f strength Gresshoff and Doy's medium supplemented with 0.5 mg/L NAA for 2 weeks and then t ransferr ing them to the same medium without any growth hormones. The rooted shoots could be grown outside the culture tube in a peat /per l i te /vermicu l i te mixture.

ABBREVIATIONS

BA: 6-Benzyl-aminopurine, GD: Gresshoff and Doy's medium, LM: conifer ce l l culture medium of Litvay et a l . , MS: modified Murashige and Skoog's medium, NAA: m-naphthaleneacetic acid.

INTRODUCTION

The internat ional demand for forest products is expected to r ise sharply over the next few decades (Thorpe and Biondi 1984). There is an urgent requirement for large numbers of improved, fast growing trees with shortened rotat ion to meet this demand. Vegetative propagation is the preferred mode of propagule production to ensure uniform forest tree populations with desirable character is t ics. However, appl icat ion of t rad i t iona l methods of vegetative propagation, such as rooting of cut t ings, to mature e l i t e trees is often d i f f i c u l t or impossible. Tissue culture has been recognized as an a l ternat ive technique for vegetative propagation (Durzan 1982). One of the possible applications of t issue culture technique for improvement of forest trees is production of mult ip le propagules from a single superior seed. Procedures for p lan t le t production have been developed for certain species including some conifers (Mott and Amerson 1981; Karnosky et a l . 1984; Franco and Schwarz 1985; Huang and Tsai 1985). However, at present no such methods are avai lable for propagation of Pinus strobus L.

Offprint requests to: K. Kaul

(white pine). The present paper describes in v i t ro p lan t le t production from explants of white pine seedlings.

MATERIALS AND METHODS

Mature white pine seeds were soaked in tap water for 16 to 18 hours, wrapped in wet cheese cloth, and then kept at 5°C in the dark for 5 to 8 weeks. Cold-treated seeds were surface s te r i l i zed (Gupta and Durzan 1985), washed 4x with s te r i l i zed double d i s t i l l e d water, and planted on germination medium of Sommer and Caldas (1981~. Seed germination was allowed to occur at 26 + i C under continuous, 2 cool white fluorescent Tl lumination (400-500 ~W/cm ). One to two week-old seedlings were used as the source of explant. The explant consisted of the top 2 to 3 mm of the hypocotyl attached to the whorl of cotyledons which had been trimmed to about hal f of the i r or ig inal length. Such an explant w i l l be referred to as the C/H (cotyledon/hypocotyl) explant. The explants were put on a modified Murashige and Skoog's medium (Kasperbauer and Reinert 1965) supplemented with growth hormones or on the medium of Litvay et a l . (1981) for induction and elongation of shoots. Root induction and elongation was achieved on a basal medium of Gresshoff and Doy (1972) modified as described in the text . At various stages of p !ant le t development cultures were incubated at 17° or 26°C. Al l media were prepared ~i th analyt ical grade reagents, gel led with 0 8% Difco I bacto-agar (unless specif ied otherwise), an d s te r i l i zed by

autoc lav ing at 121°C and 1 kg/cm ~ pressure for 15 min. The pH of a l l media was adjusted to 5.8 before s t e r i l i z a t i o n . Al l experiments were repeated at least twice.

1Any opinions, f indings, conclusions, and/or recommendations expressed in this publ icat ion are those of the author and do not necessarily re f lec t the view of the United States Department of Agr icul ture, Cooperative State Research Service (USDA/CSRS). Mention of a trade name does not const i tute a guarantee or warranty of the product by USDA/CSRS and does not imply the i r approval to the exclusion of other products that may also be sui table.

RESULTS AND DISCUSSION

Plant le t development was achieved in four discrete steps - shoot induct ion, shoot growth, root induct ion, and root growth.

Shoot induct ion

Shoot induction in C/H explants occurred when the explants were planted on MS supplemented with 2.0 mg/L BA and 0.2 mg/L NAA (medium C) and incubated at 26 + l°C under i l l umina t ion (see Materials and Methods). Af ter 2 weeks in cul ture the hypocotyl port ion of the explant became swollen and showed some cal lus formation at the basal end. No adventi t ious shoots were v i s i b le at th is time. Omission of NAA from the medium resulted only in poor shoot induct ion. An induction period of 2 to 3 weeks gave the best resul ts . Extending the length of cul ture period on induction medium to 5 or 6 weeks resulted in extensive ca l lus ing from the hypocotyl and a reduction in the number of shoots formed. Continuous cul ture on medium C caused extensive ca l lus ing wi thout the growth of any shoots. Shoot induct ion was an essential step. No advent i t ious shoots were formed i f C/H explants were put d i rec t l y on the shoot growth medium.

Shoot growth

For shoot growth C/H explants were transferred from shqot induct ion medium to LM medium (Li tvay et a l . 1981) wi thout any plant growth hormones. L ight and temperature condit ions for shoot growth were the same as those for shoot induct ion. Adventi t ious shoots became v i s i b le a f ter about a week on LM medium. New advent i t ious shoots continued to be formed during the next 6 to 8 weeks. Induction on medium C for 2 weeks fol lwed by t ransfer to LM resulted in advent i t ious shoot formation in 100% of the C/H explants. The advent i t ious shoots resulted from the growth of buds formed in the ax i ls of cotyledons during the shoot induct ion phase. No bud formation and subsequent shoot growth occurred when segments of hypocotyl and cotyledons were cultured on medium C for 2 weeks and then transferred to LM medium. The number of adventi t ious shoots per cul ture varied between 8 and 21 with an average of 14.7 shoots per cul ture (Fig. 1).

These shoots could be eas i ly pul led apart from each other. They ranged from 3 to 7 mm in size. I f NAA was omitted from the induction medium the number of advent i t ious shoots decreased remarkably - the number varied from I to 4 shoots per cul ture. These shoots were very hard to separate from each other without in ju ry . Since the seeds used in the present study were col lected from open pol l inated trees, i t is possible that some of the v a r i a b i l i t y in the number of advent i t ious shoots produced is due to genetic di f ferences.

Root induct ion

Shoots from LM medium were transferred to a ha l f strength G & D medium (Gresshoff and Doy 1972) containing i0 mg/L i n o s i t o l , 1% sucrose, 1% agar, and 0.5 mg/L NAA, for 2 weeks under l i g h t and temperature condit ions used for shoot induct ion and development.

Root 9rowth

Af ter 2 weeks shoots from root induct ion medium were transferred to root growth medium which was s~milar to root induction medium but without any growth hormones. Cultures were incubated under 150-200 ~W/cm ~ ~luorescent i l l umina t ion (16-hour days) at 17 + ivC. One or two roots appeared in some c u l t u r e s w i t h i n a week of t ransfer to root growth medium. Root growth occurred in about 80% of the cultures w i th in 5 weeks (Fig. 2). Root induction and growth has also been achieved in advent i t ious shoots of Lar ix decidua and Pinus banksiana, produced in---vTtro, by using a very s im i la r media combination (Karnosky et a l . 1984).

FIG. 2. A p lan t l e t with roots (r) 5 weeks a f ter being transferred to root growth medium.

FIG. i . Production of mul t ip le shoots from a C/H explant on LM medium preceded by 2 weeks incubation on medium C.

Growth of in ~ i t ro produced p lan t l e t s

Plant lets with 1 cm or longer roots were transferred to a so i l - l ess mixture - pea t /pe r l i t e / vermicu l i te , i /2 /1 - used by Karnosky et a l . (1984) and grown under high humidity (Fig. 3). Af ter about 4 weeks these plants can be grown under normal (40-50%) humidity. Some of the plants have now been grown for four months outside the cul ture tube. To date a tota l of 138 plants have been obtained. At present a period of 16 to 25 weeks is required for obtaining rooted plants from C/H explants.

FIG. 3. In v i t ro produced plant let 4 weeks after transfer to peat/per l i te/vermicul i te, 1/2/1.

ACKNOWLEDGEMENTS

This research was supported by USDA/CSRS Grant No. KYX10-85-O3P. The technical assistance of Linda Winkle is grateful ly acknowledged. I thank Mr. Newton Coop for the seeds used in this study. I thank Dr. Harold R. Benson, Director of Land-Grant Programs, and his s taf f for their continuous support.

REFERENCES

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Franco EO, Schwarz OJ (1985) In: Henke RR, Hughes KW, Constantin MJ, Hollaender A (eds) Tissue Culture in Forestry and Agriculture. Plenum Press, New York, pp 195-213.

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