Plant Cell Reports (1985) 4:180-183

Plant Cell Reports © Springer-Verlag 1985

Growth and differentiation of callus cultures of Pinus

Karan Kaul and T. S. Kochhar

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

Received November 5, 1984 / Revised version received May 14, 1985 - Communicated by I. K. Vasil

ABSTRACT

Segraents of hypoeotyl and cotyledons of aseptically-grown seedlings of Pinus strobus L. (white pine) and P. echinata Mill (shortleaf pine) were used as explants for establishing tissue cultures. Growth and differentiation of callus were studied on a modified Murashige and Skoog's medium containing nutrients and plant growth regulators. Meristems below the surface of callus tissue of P. strobus could be induced on media supplemented with ~-naphthaleneacetic acid alone or in combination with certain other plant growth regulators. Occasionally, differentiation of shoot buds also occurred on callus cultures. These shoot buds could be grown in vitro but roots did not develop.

ABBREVI ATI ONS

ABA: abscisic acid, BA: 6-Benzyl-arninopurine, 2-ip: N6-(A2-isopentanyl)-adenine, GD: Gresshoff and Doy's medium, GE: Gamborg and Eveleigh's medium, MS: Modified Murashige and Skoog's medium, NAA: ~-naphthaleneacetic acid, SC: Son, her and Caldas' medium, TIBA: 2,3,5-Triiodobenzoic acid.

INTRODUCTION

Vegetative propagation of conifers old enough to have demonstrated their superior characteristics by rooting of cuttings is often difficult. It is therefore important to develop an alternative method of rapid vegetative propagation to mass produce trees which have desirable characteristics.

The potential of plant tissue culture technique as a means of mass propagation of superior forest trees including conifers has been recognized for several years (Bonga 1974; Durzan and Campbell 1974 ; Konar and Nagmani ~ 1974 ; Sonmer and Brown 1979; Karnosky 1981; Mort 1981). However, this potential remains largely unexploited, primarily because of our insufficient knowledge of the in-vitro physiology of ~tissues from conifers (Durzan 1982). In-vitro regeneration of plantlets from primary explants of several gymnosperm species has .been ~ reported (Sommer et al : 1975 ; Cheng 1975 ; Mott et- al< 1977; Reil~ly and'Washer 1977; Arnoid and Eriksson 1978, '1981; Minocha 1980;--David et-al. 1982). However, plantlet differentiation from callus cultures has not been demonstrated (Thorpe and Biondi 1984). Development of methodology to regenerate plantlets from callus is essential

before techniques such as genetic engineering and somatic hybridization can be used for introduction of desirable characteristics in coniferous forest trees. Detailed knowledge of nutritional and hormonal requirements for callus proliferation, its growth, and its subsequent differentiation to form plantlets is necessary for each individual species before the mass in-vitro propagation can become a reality. The present conmunication describes callus establishment from Pinus strobus and P. echinata. A limited amount of differentiation, and shoot regeneration from Pinus strobus is also being reported.

MATERIALS AND METHODS

Plant Materials and Seedling Production

Experiments were done using two species of Pinus - P. strobus L. (white pine) and P. echinata Mill (shortleaf pine). Seeds were obtained from the Kentucky Division of Forestry (white and short-leaf); National tree seed laboratory, Dry Branch, GA (white); and F. W. Schumacher Co., Sandwich, MA (white). Seeds were soaked in tap water for 18 hours, wrapped in wet cheese cloth, and stored at 5°C in dark for 5 to 8 weeks. Cold- treated seeds were surface-sterilized in 50% clorox I (2.625% sodium hypochlorite) for 15 rain, washed 4 times with sterilized double-distilled water, and planted on modified Risser and White's medium (Sommer and Caldas 1981) without any growth hormones. Germination was allowed to occur at 26+IOC under 450-500 zW/cm 2 cool white fluorescent illumination (16-hour days).

Establishment of Callus Cultures

Segments (3 to 6 n~n) of cotyledons and hypocotyl from 4 to 6 week-old aseptically-grown

iAny opinions, findings, conclusions, and/or recommendations expressed in this publication are those of the authors and do not necessarily reflect the view of the United States Department of Agriculture, Cooperative State Research Service (USDA/CSRS). Mention of a trade name does not constitute a guarantee or warranty of the product by USDA/CSRS and does not imply their approval to the exclusion of other products that may also be suitable.

I

seedlings were placed on a modified (Kasperbauer and Reinert 1965) Murashige and Skoog's medium (MS) supplemented with 0.2 mg/L NAA and 2 mg/L BA. All media used in the present study were prepared with analytical grade reagents and were sterilized by autoclaving at 1 kg/~n 2 pressure at 121°C for 15 rain. ABA was filter sterilized before it was added to autoclaved medium. The pH of all media was adjusted to 5.8 before sterilizing. Tissue cultures were growl either on i0 ml medium in 25x150 n]n culture tubes or on 20 ml medium in 125 ml Erlenmeyer flasks. In all experiments 40 to S0 mg pieces of callus which has been subcultured 1 to S-times after initial proliferation on the primary explant, were used as inoeulum. This corresponded to approximately 5 mg dry weight. All cultures were maintained at 26+I°C under 450-500 ~W/cm 2 cool white fluorescent illu~ination (16-hour days). Dry weight determinations were done after drying the tissue at @0°C for 48 hr.

For anatomical studies tissue was fixed in formaldehyde/acetic acid/95% ethanol, 90:5:5 (v/v) for 24 hr and then stored in 70% ethanol at room temperature. An ethanol/xylene series was used for dehydration. Material was embedded in paraffin. Twelve micron thick sections were cut. These were stained with safranin and fast green.

In each experiment I0 to 20 cultures were used. Each experiment was repeated at least once.

RESULTS AND DISCUSSION

Establishment and Maintenance of Tissue Cultures

Callus proliferation and gro~Ngh were studied on GD, SC, MS, and GE media supplemented with 0.2 mg/L NAA and 2 mg/L BA. Best callus proliferation occurred on MS + 0.2 mg/L NAA + 2 mg/L BA (medium C). Experiments were done to compare callus proliferation on medium C from explants of seedlings derived from seeds of several different populations of white pine collected from New England, New York, and Virginia in U.S.A. and from Yugoslavia. There were minor differences in callusing responses between the explmDts of various groups. Medium C has also been found to be excellent for the maintenance of the callus tissue derived from all the explants.

m

~ l t e Pfne C~11us

i B Shortleaf Pine Callus

m

m ~ ~ E ~ - - - E i E

1 2 3 4 5 6

NUNBER OF SUBCULTURE

FIG. i Increase in dry weight of callus tissue after subeulturing at 5-week intervals on medium C. Increase has been shown as "fold of the dry weight of inoeulum". White pine callus showed a 9 to 10-fold increase throughout the course of the experiment whereas shortleaf pine callns showed only 4 to 5-fold increase after the third subculture.

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The growth patterns of white and shortleaf pine calli were quite different when they were repeatedly subcultured at S-week intervals on medium C. For the first two subcultures, after initiation of callus from the primary explant, dry weight of both kinds of ealli increased about 10-fold after 5 weeks (Fig. I). ~nite pine c~llus continued to show a $ to 10-fold increase in the subsequent subculture (Fig. I). Shortleaf pine callus on the other hand showed only about S-fold increase from the third subculture onwards (Fig. I). White pine callus remained dark green and "healthy- looking" througbout the experiment. But, shortleaf pine callus cultures contained patches of brown necrotic tissue after the third subculture.

Both half strength and full strength medium C supported callus growth. Media containing one eighth, one fourth, and double strength mineral and vitamins of medium C in combination with 2% sucrose, i00 mg/L inositol, 0.2 my/L NAA, and 2 mg/L BA were somewhat less effective (Fig. 2).

80 i

60 m

40 m,,

20 m

0 m,

I I I I O m O Whtte Ptne Callus mm

~ m O Short leaf Ptne Callus

i n n I i ~ 1 / 8 ~ 1 / 4 CX1/2 C Cx2

CONC. OF H]NE~LS AND V ] T ~ I H S

FIG. 2 Effect of mineral and vitamin concentration on growth of callus tissue of white and shortleaf pine. Good growth was supported by half as well as full strength mineral and vitamins in combination with 2% sucrose, I00 mg/L inositol, 0.2 mg/L NAA, and 2 mg/L BA.

Effects of Inositol Concentration on Callus Growth

~%~ite pine callus required some inositol for growth. In absence of inositol white pine tissue turned brown and necrotic. Under the experimental conditions used, 50 mg/L inositol gave optimal callus growth. Concentrations higher than 50 mg/L inhibited callus growth slightly (Fig. 3). On S0, 100, and 200 mg/L inositol white pine tissue was dark green, nonfriable and compact. However, on media with 10, 25, or 400 mg/L inositol white pine callus was pale green or pinkish and friable. The best growth of shortleaf pine callus occurred in absence of any inositol in the medium (Fig. 3)

Effects of Plant Growth HoiTnones on Callus Growth and Differentiation

In an attempt to induce organogenesis in the callus cultures a number of plant growth hormones in various combinations were added to ~he cul~ure media.

Experiments were done using 0, 0.01, 0.I, 0.2, and 1.0 mg/L NAA in combination with 0, 0.I,~0.2, 1.0, 2.0, and I0.0 mg/L BA. On MS containing 0.I or 0.2 mg/L NAA alone or in combination with 0.I or 0.2 mg/L BA, meristematic areas appeared in the

182

I00

80

60

40

20

~ m ~ Wh|te Ptne Callus " ~rlL ~ L Shortleaf Ptne Callus

~ o

__&

0 100 200 300 400

MG ]NOSITOL PER L MEDIUM

FIG. 3 Effect of inositol concentration in medium C on growth of white and shortleaf pine calli. }'~nite pine calltk~ did not grow in absence of inositol. Fifty mg/L inositol was optimal for the growth of white pine callus. Growth of shortleaf pine callus was best in absence of any inositol and was inhibited by all concentrations of inositol used in the present study.

callus (Figs. 4, 5) 2 to 4 weeks after inoculation. Five to six weeks after inoculation 100% of white pine callus cultures became very dark green and had many protuberances on the surface, which probably resulted from meristematic activity just below the surface of callus tissue (Fig. 6). Although these protuberances persisted for 14 to 16 weeks (after which time the cultures were discarded), they did not grow any further. Small portions of callus bearing these protuberances were transferred to MS + 0.2 mg/L NAA and MS or Modified Risser and White's medium without any growth hormones. No further development occurred on any of the media used. No protuberances were formed on the surface when white pine callus was grown on MS medium supplemented with 0. I or 0.2 mg/L NAA in combination with 0.i or 0.2 mg/L 2-ip.

In order to determine whether a cytokinin other than BA would be a more effective inducer of differentiation, MS media containing 0.I mg/L of NAA in combination with kinetin, 2-ip, zeatin, and zeatin riboside were tried. In all instances the amount of cytokinin added was equimolar to 0. I mg/L BA. Only media containing NAA in combination with kinetin or zeatin riboside induced the formation of meristematic protuberances The extent of meristem differentiation was no more than that on BA/NAA media.

Minocha (1980) reported shoot formation on P. strobus embyros cultured on media supplemented with TIBA. In the present study effect of TIBA on differentiation in P. strobus callus was examined. Callus tissue was grown on the following media containing TIBA: MS + 1 mg/L TIBA, MS + 0. I mg/L BA + 0.i mg/L NAA + 1 mg/L TIBA, and MS + 0.2 mg/L NAA + 0.i, 0.5, i, 2, and 5 mg/L TIBA. None of these media induced differentiation in callus cultures, Our results with callus tissue are different from those reported for white pine embryonic tissue (Minocha 1980). These differences may be due to genetic differences in the material used, or perhaps to physiological differences between embryonic tissue and callus derived from seedling explants.

FIGS. 4-7 Anatomical view of differentiation Jn white pine tissue cultures. 4, 5: Meristematic areas below the surface of callus grown on ~ + 0.2 mg/L NAA - 28 days after inoculation. In Fig. 5 notice the cells in division (arrows) and a structure reminiscent of a young embryo(e). @: Cross section of a surface protuberance on callus grown on MS + 0.2 mg/L NAA - 70 days after inoculation. 7: Cross section of 35-day old callus culture grown on i/2 MS + 50 mg/L inositol + 0.2 mg/L BA showing shoot bud (Sb) differentiation. The shoot bud has differentiated from the callus tissue and not from a leftover portion of the primary explant. Bar = 95 pm in Fig. 4, 65 ~m in Fig. 5, 215 ~m in Fig. 6, and 1200 ~m in Fig. 7.

Abscisie acid (ABA) has been shown to induce organogenesis in tissue cultures of certain angiosperms (Shepard 1980). Media supplemented with ABA were therefore tried for induction of org~ogenesis in P. strobus callus cultures. When provided alone in a concentration range of 0.02 to 2.0 mg/L ABA did not induce any organogenesis. Growth of tissue was markedly retarded on media containing 0.2 mg/L or more of ABA. However, good callus growth occurred on MS + 0.2 mg/L NAA + 0.5 mg/L zeatin + 0.02 to 0.2 mg/L ABA. Differentiation of subsurface meristems was induced on media containing 0.02 mg/L ABA along with 0.2 mg/L NAA and 0.5 mg/L zeatin.

Occasional differentiation of shoot buds occurred on the half strength MS + 50 mg/L inositol + 0.2 mg/L NAA + 2 mg/L BA and MS + 0.I mg/L NAA + 0.156 mg/L zeatin riboside. Unlike the protuberance differentiation, shoot differentiation was inconsistent and occurred in 0 to 30% of the callus cultures. Six to I0 shoots differentiated in a culture. White pine cultures with shoot buds are shown in Figs. 7 and 8. In cross sectional view (Fig. 7) two shoot buds can be clearly seen to have differentiated from the callus and not from any remnants of the primary explant. Such shoot buds grew to a size of 5-15 rrm. These could be transplanted on fresh medium. On modified Risser and ~hite's medium (Sommer and Caldas 1981) without any growth regulators or on the same medium supplemented with 0.05 mg/L NAA and 2 mg/L IBA, no rooting could be induced (Fig. 9). Addition of 1% activated charcoal to m~y of the above media also failed to induce rooting.

FIGS. 8, 9 Organogenesis in white pine callus cultures. 8: Shoot buds (Sb) on an 8-week-old callus culture grown on MS + 50 mg/L inositol + 0.2 mg/L NAA + 2 mg/L BA. 9: Shoot obtained from callus grown on MS + 0.1 mg/L NAA + 0.156 mg/L zeatin riboside. No rooting could be induced on such shoots.

Media which gave a limited degree of differentiation in white pine callus induced prolific callus growth but no differentiation in shortleaf pine callus.

Although differentiation of buds from subcultured white pine callus occurred only sporadically in our experiments, it nevertheless indicates that some cells in seedling-derived

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callus of white pine have the regenerative potential. It is hoped that future research will define the in-vitro conditions under which a larger proportion of cells can be induced to. express this potential.

ACKNOWLEDGE~Z~fS

This research was supported by USDA/CSRS Grant No. KYXI282000008. We thank Dr. M. J. Kasperbauer for his input during numerous discussions regarding this research. We thank Mr. David Fisher and Mr. Charles Kelly of the Kentucky Division of Forestry for providing us pine seeds. The assistance of Linda Winkle, Michael Holman, R. Todd Haramons, and Daniel Yancey is gratefully acknowledged. We thank Dr. Lionel Williarr~on, Research Director of KSUCRS and his staff for their continuous support.

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