Plant Cell Reports
Plant Cell Reports (1987) 6:5-7
© 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 adventitious shoots were obtained from cotyledon-hypocotyl explants of a s e p t i c a l l y 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 h a l f of t h e i r original 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 Litvay et al. without any growth hormones for 8 to i0 weeks to obtain shoot induction and shoot growth. Root induction and root growth were achieved by culturing the shoots on h a l f strength Gresshoff and Doy's medium supplemented with 0.5 mg/L NAA for 2 weeks and then transferring them to the same medium without any growth hormones. The rooted shoots could be grown outside the culture tube in a p e a t / p e r l i t e / v e r m i c u l i t e mixture. ABBREVIATIONS BA: 6-Benzyl-aminopurine, GD: Gresshoff and Doy's medium, LM: conifer c e l l culture medium of Litvay et a l . , MS: modified Murashige and Skoog's medium, NAA: m-naphthaleneacetic acid. INTRODUCTION The international demand for forest products is expected to rise 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 rotation to meet this demand. Vegetative propagation is the preferred mode of propagule production to ensure uniform forest tree populations with desirable c h a r a c t e r i s t i c s . However, application of t r a d i t i o n a l methods of vegetative propagation, such as rooting of cuttings, 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 t e r n a t i v e technique for vegetative propagation (Durzan 1982). One of the possible applications of tissue culture technique for improvement of forest trees is production of multiple propagules from a single superior seed. Procedures for p l a n t l e t production have been developed for certain species including some conifers (Mott and Amerson 1981; Karnosky et al. 1984; Franco and Schwarz 1985; Huang and Tsai 1985). However, at present no such methods are available for propagation of Pinus strobus L. Offprint requests to: K. Kaul
(white pine). The present paper describes in v i t r o p l a n t l e 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 f o r 5 to 8 weeks. Cold-treated seeds were surface s t e r i l i z e d (Gupta and Durzan 1985), washed 4x with s t e r i l i z e d 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 Tllumination (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 h a l f of t h e i r original 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 al. (1981) f o r 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 t e x t . At various stages of p ! a n t l e t development cultures were incubated at 17° or 26°C. All media were prepared ~ith analytical grade reagents, gelled with 0 8% Difco I bacto-agar (unless specified otherwise), and s t e r i l i z e d by a u t o c l a v i n g 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 . All experiments were repeated at least twice.
1Any opinions, findings, conclusions, and/or recommendations expressed in this publication are those of the author and do not necessarily r e f l e c t 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 t h e i r approval to the exclusion of other products that may also be suitable.
RESULTS AND DISCUSSION P l a n t l e t development was achieved in four discrete steps - shoot induction, shoot growth, root induction, and root growth. Shoot induction 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 u m i n a t i o n (see Materials and Methods). A f t e r 2 weeks in culture the hypocotyl portion of the explant became swollen and showed some callus formation at the basal end. No adventitious shoots were v i s i b l e at t h i s time. Omission of NAA from the medium resulted only in poor shoot induction. An induction period of 2 to 3 weeks gave the best r e s u l t s . Extending the length of culture period on induction medium to 5 or 6 weeks resulted in extensive c a l l u s i n g from the hypocotyl and a reduction in the number of shoots formed. Continuous culture on medium C caused extensive c a l l u s i n g without the growth of any shoots. Shoot induction was an essential step. No adventitious shoots were formed i f C/H explants were put d i r e c t l y on the shoot growth medium. Shoot growth For shoot growth C/H explants were transferred from shqot induction medium to LM medium (Litvay et al. 1981) without any plant growth hormones. Light and temperature conditions f o r shoot growth were the same as those for shoot induction. Adventitious shoots became v i s i b l e a f t e r about a week on LM medium. New adventitious shoots continued to be formed during the next 6 to 8 weeks. Induction on medium C f o r 2 weeks follwed by t r a n s f e r to LM resulted in adventitious shoot formation in 100% of the C/H explants. The adventitious shoots resulted from the growth of buds formed in the a x i l s of cotyledons during the shoot induction phase. No bud formation and subsequent shoot growth occurred when segments of hypocotyl and cotyledons were cultured on medium C f o r 2 weeks and then transferred to LM medium. The number of adventitious shoots per culture varied between 8 and 21 with an average of 14.7 shoots per culture (Fig. 1).
These shoots could be e a s i l y pulled 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 adventitious shoots decreased remarkably - the number varied from I to 4 shoots per culture. These shoots were very hard to separate from each other without i n j u r y . Since the seeds used in the present study were collected from open p o l l i n a t e d trees, i t is possible that some of the v a r i a b i l i t y in the number of adventitious shoots produced is due to genetic differences. Root induction Shoots from LM medium were transferred to a h a 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 conditions used f o r shoot induction and development. Root 9rowth A f t e r 2 weeks shoots from root induction 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 u m i n a t i o n (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 transfer to root growth medium. Root growth occurred in about 80% of the cultures w i t h i n 5 weeks (Fig. 2). Root induction and growth has also been achieved in adventitious shoots of Larix decidua and Pinus banksiana, produced in---vTtro, by using a very s i m i l a r media combination (Karnosky et al. 1984).
FIG. 2.
A p l a n t l e t with roots (r) 5 weeks a f t e r being transferred to root growth medium.
Growth of in ~ i t r o produced p l a n t l e t s
FIG. i .
Production of m u l t i p l e shoots from a C/H explant on LM medium preceded by 2 weeks incubation on medium C.
Plantlets with 1 cm or longer roots were transferred to a s o i l - l e s s mixture - p e a t / p e r l i t e / v e r m i c u l i t e , i / 2 / 1 - used by Karnosky et al. (1984) and grown under high humidity (Fig. 3). A f t e r 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 culture tube. To date a t o t a l of 138 plants have been obtained. At present a period of 16 to 25 weeks is required f o r obtaining rooted plants from C/H explants.
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. Gresshoff PM, Doy CH (1972) Planta 107:161-170. Gupta PK, Durzan DJ (1985) Plant Cell Reports 4:177-179. Huan9 FH, Tsai H (1985) In: Henke RR, Hughes KW, Constantin MJ, Hollaender A (eds) Tissue Culture in Forestry and Agriculture, Plenum Press, New York, p 326.
FIG. 3.
In vitro produced plantlet 4 weeks after transfer to peat/perlite/vermiculite, 1/2/1.
Karnosky DF, Diner AM, Mickler RA (1984) In: Hanover J, Karnosky D, KeathleyD (eds) Proceedings International Symposium of Recent Advances in Forest Biotechnology, Michigan Biotechnology I n s t i t u t e , East Lansing, Michigan, pp. 69-73. Litvay JD, Johnson MA, Verma D, Einspahr D, Weyrauch K (1981) Institute of Paper Chemistry Technical Paper Series No. 115.
ACKNOWLEDGEMENTS This research was supported by USDA/CSRSGrant No. KYX10-85-O3P. The technical assistance of Linda Winkle is gratefully 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 t a f f for their continuous support.
Sommer HE, Caldas LS (1981) In: Thorpe, TA (ed) Plant Tissue Culture Methods and Applications in Agriculture, Academic Press, New York, pp 349-358.
REFERENCES Durzan DJ (1982) In: Bonga JM, Durzan DJ (eds) Tissue Culture in Forestry, Nijhoff/Junk Boston, pp 36-71.
Thorpe TA, Biondi S (1984) In: Sharpe WR, Evans DA, Anunirato PA, Yamada Y (eds) Handbook of Plant Cell Culture, vol. 2, Crop Species, Macmillan Publishing Company, New York, pp 435-470.
Mott RL, Amerson HV (1981) Tech. Bull. No. 271 of North Carolina Agricultural Research Service.
Plant Cell Reports (1987) 6:5-7
© 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 adventitious shoots were obtained from cotyledon-hypocotyl explants of a s e p t i c a l l y 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 h a l f of t h e i r original 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 Litvay et al. without any growth hormones for 8 to i0 weeks to obtain shoot induction and shoot growth. Root induction and root growth were achieved by culturing the shoots on h a l f strength Gresshoff and Doy's medium supplemented with 0.5 mg/L NAA for 2 weeks and then transferring them to the same medium without any growth hormones. The rooted shoots could be grown outside the culture tube in a p e a t / p e r l i t e / v e r m i c u l i t e mixture. ABBREVIATIONS BA: 6-Benzyl-aminopurine, GD: Gresshoff and Doy's medium, LM: conifer c e l l culture medium of Litvay et a l . , MS: modified Murashige and Skoog's medium, NAA: m-naphthaleneacetic acid. INTRODUCTION The international demand for forest products is expected to rise 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 rotation to meet this demand. Vegetative propagation is the preferred mode of propagule production to ensure uniform forest tree populations with desirable c h a r a c t e r i s t i c s . However, application of t r a d i t i o n a l methods of vegetative propagation, such as rooting of cuttings, 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 t e r n a t i v e technique for vegetative propagation (Durzan 1982). One of the possible applications of tissue culture technique for improvement of forest trees is production of multiple propagules from a single superior seed. Procedures for p l a n t l e t production have been developed for certain species including some conifers (Mott and Amerson 1981; Karnosky et al. 1984; Franco and Schwarz 1985; Huang and Tsai 1985). However, at present no such methods are available for propagation of Pinus strobus L. Offprint requests to: K. Kaul
(white pine). The present paper describes in v i t r o p l a n t l e 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 f o r 5 to 8 weeks. Cold-treated seeds were surface s t e r i l i z e d (Gupta and Durzan 1985), washed 4x with s t e r i l i z e d 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 Tllumination (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 h a l f of t h e i r original 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 al. (1981) f o r 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 t e x t . At various stages of p ! a n t l e t development cultures were incubated at 17° or 26°C. All media were prepared ~ith analytical grade reagents, gelled with 0 8% Difco I bacto-agar (unless specified otherwise), and s t e r i l i z e d by a u t o c l a v i n g 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 . All experiments were repeated at least twice.
1Any opinions, findings, conclusions, and/or recommendations expressed in this publication are those of the author and do not necessarily r e f l e c t 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 t h e i r approval to the exclusion of other products that may also be suitable.
RESULTS AND DISCUSSION P l a n t l e t development was achieved in four discrete steps - shoot induction, shoot growth, root induction, and root growth. Shoot induction 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 u m i n a t i o n (see Materials and Methods). A f t e r 2 weeks in culture the hypocotyl portion of the explant became swollen and showed some callus formation at the basal end. No adventitious shoots were v i s i b l e at t h i s time. Omission of NAA from the medium resulted only in poor shoot induction. An induction period of 2 to 3 weeks gave the best r e s u l t s . Extending the length of culture period on induction medium to 5 or 6 weeks resulted in extensive c a l l u s i n g from the hypocotyl and a reduction in the number of shoots formed. Continuous culture on medium C caused extensive c a l l u s i n g without the growth of any shoots. Shoot induction was an essential step. No adventitious shoots were formed i f C/H explants were put d i r e c t l y on the shoot growth medium. Shoot growth For shoot growth C/H explants were transferred from shqot induction medium to LM medium (Litvay et al. 1981) without any plant growth hormones. Light and temperature conditions f o r shoot growth were the same as those for shoot induction. Adventitious shoots became v i s i b l e a f t e r about a week on LM medium. New adventitious shoots continued to be formed during the next 6 to 8 weeks. Induction on medium C f o r 2 weeks follwed by t r a n s f e r to LM resulted in adventitious shoot formation in 100% of the C/H explants. The adventitious shoots resulted from the growth of buds formed in the a x i l s of cotyledons during the shoot induction phase. No bud formation and subsequent shoot growth occurred when segments of hypocotyl and cotyledons were cultured on medium C f o r 2 weeks and then transferred to LM medium. The number of adventitious shoots per culture varied between 8 and 21 with an average of 14.7 shoots per culture (Fig. 1).
These shoots could be e a s i l y pulled 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 adventitious shoots decreased remarkably - the number varied from I to 4 shoots per culture. These shoots were very hard to separate from each other without i n j u r y . Since the seeds used in the present study were collected from open p o l l i n a t e d trees, i t is possible that some of the v a r i a b i l i t y in the number of adventitious shoots produced is due to genetic differences. Root induction Shoots from LM medium were transferred to a h a 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 conditions used f o r shoot induction and development. Root 9rowth A f t e r 2 weeks shoots from root induction 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 u m i n a t i o n (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 transfer to root growth medium. Root growth occurred in about 80% of the cultures w i t h i n 5 weeks (Fig. 2). Root induction and growth has also been achieved in adventitious shoots of Larix decidua and Pinus banksiana, produced in---vTtro, by using a very s i m i l a r media combination (Karnosky et al. 1984).
FIG. 2.
A p l a n t l e t with roots (r) 5 weeks a f t e r being transferred to root growth medium.
Growth of in ~ i t r o produced p l a n t l e t s
FIG. i .
Production of m u l t i p l e shoots from a C/H explant on LM medium preceded by 2 weeks incubation on medium C.
Plantlets with 1 cm or longer roots were transferred to a s o i l - l e s s mixture - p e a t / p e r l i t e / v e r m i c u l i t e , i / 2 / 1 - used by Karnosky et al. (1984) and grown under high humidity (Fig. 3). A f t e r 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 culture tube. To date a t o t a l of 138 plants have been obtained. At present a period of 16 to 25 weeks is required f o r obtaining rooted plants from C/H explants.
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. Gresshoff PM, Doy CH (1972) Planta 107:161-170. Gupta PK, Durzan DJ (1985) Plant Cell Reports 4:177-179. Huan9 FH, Tsai H (1985) In: Henke RR, Hughes KW, Constantin MJ, Hollaender A (eds) Tissue Culture in Forestry and Agriculture, Plenum Press, New York, p 326.
FIG. 3.
In vitro produced plantlet 4 weeks after transfer to peat/perlite/vermiculite, 1/2/1.
Karnosky DF, Diner AM, Mickler RA (1984) In: Hanover J, Karnosky D, KeathleyD (eds) Proceedings International Symposium of Recent Advances in Forest Biotechnology, Michigan Biotechnology I n s t i t u t e , East Lansing, Michigan, pp. 69-73. Litvay JD, Johnson MA, Verma D, Einspahr D, Weyrauch K (1981) Institute of Paper Chemistry Technical Paper Series No. 115.
ACKNOWLEDGEMENTS This research was supported by USDA/CSRSGrant No. KYX10-85-O3P. The technical assistance of Linda Winkle is gratefully 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 t a f f for their continuous support.
Sommer HE, Caldas LS (1981) In: Thorpe, TA (ed) Plant Tissue Culture Methods and Applications in Agriculture, Academic Press, New York, pp 349-358.
REFERENCES Durzan DJ (1982) In: Bonga JM, Durzan DJ (eds) Tissue Culture in Forestry, Nijhoff/Junk Boston, pp 36-71.
Thorpe TA, Biondi S (1984) In: Sharpe WR, Evans DA, Anunirato PA, Yamada Y (eds) Handbook of Plant Cell Culture, vol. 2, Crop Species, Macmillan Publishing Company, New York, pp 435-470.
Mott RL, Amerson HV (1981) Tech. Bull. No. 271 of North Carolina Agricultural Research Service.
