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A protocol is described for somatic embryogenesis (SE) Pinus elliottii × P. caribaea, an interspecific pine hybrid of major economic importance. Different embryogenesis conditions were tested, from time of initiation to plant regeneration. Immature megagametophytes containing zygotic embryos of five open pollinated (OP) mother trees were used for SE initiation. An established rate of 10.2% was achieved for explants inoculated on modified Litvay's medium (mLV), supplemented with BAP (4.5 µM) and 2,4-D (9 µM). Initiation of SE was influenced by the mother-tree genotype however, modification of culture conditions enabled establishment of embryogenic cell lines (ECLs) from all OP seed families. Fifty-two percent of somatic embryos developed to the cotyledonary stage on mLV, supplemented with 40 µM ABA and 6% w/v sucrose. Cotyledonary stage somatic embryos converted to plants at 45-86%. Four months after acclimatization in a greenhouse, plant survival rates ranged between 78 and 94%. Flow cytometry analysis of ploidy, did not show any variation in ECLs and somatic seedlings. [ABSTRACT FROM AUTHOR]

An efficient protocol was developed for large-scale micropropagation of slash pine (Pinus elliottii Engelm. var. elliottii). For that, explants consisting of shoot apices 1.5–2.0 cm in length from 4 wk-old seedlings were cultured on two different basal media (Murashige & Skoog (MS) and Westvaco WV5 (WV5)). All media were supplemented with 6-benzylaminopurine (BAP), to stimulate the formation of axillary buds. Best bud induction was achieved, after 4 wk, on Westvaco WV5 medium (with 10-μM BAP) with rates close to 100%, and an average number of ~ 7 new buds formed per explant. Elongation took place for 6 wk on Westvaco WV5 medium containing activated charcoal (0.2% (w/v)) and without growth regulators. Rooting took place on half-strength Westvaco WV5 medium containing 9.8-μM indole-3-butyric acid (IBA). After 6 wk, root primordia were visible in ~ 43% of shoots. The acclimatization protocol was also optimized by controlling relative humidity, light/photoperiod, temperature, and nutrition, which led to an acclimatization success of ~ 89%. Flow cytometry analysis of DNA-ploidy did not show any variation between micropropagated plants and seedlings. With the protocol here described, it is possible to obtain a high number of genetically uniform plants per explant, 1 yr after in vitro germination of slash pine seeds.An efficient protocol was developed for large-scale micropropagation of slash pine (Pinus elliottii Engelm. var. elliottii). For that, explants consisting of shoot apices 1.5–2.0 cm in length from 4 wk-old seedlings were cultured on two different basal media (Murashige & Skoog (MS) and Westvaco WV5 (WV5)). All media were supplemented with 6-benzylaminopurine (BAP), to stimulate the formation of axillary buds. Best bud induction was achieved, after 4 wk, on Westvaco WV5 medium (with 10-μM BAP) with rates close to 100%, and an average number of ~ 7 new buds formed per explant. Elongation took place for 6 wk on Westvaco WV5 medium containing activated charcoal (0.2% (w/v)) and without growth regulators. Rooting took place on half-strength Westvaco WV5 medium containing 9.8-μM indole-3-butyric acid (IBA). After 6 wk, root primordia were visible in ~ 43% of shoots. The acclimatization protocol was also optimized by controlling relative humidity, light/photoperiod, temperature, and nutrition, which led to an acclimatization success of ~ 89%. Flow cytometry analysis of DNA-ploidy did not show any variation between micropropagated plants and seedlings. With the protocol here described, it is possible to obtain a high number of genetically uniform plants per explant, 1 yr after in vitro germination of slash pine seeds.An efficient protocol was developed for large-scale micropropagation of slash pine (Pinus elliottii Engelm. var. elliottii). For that, explants consisting of shoot apices 1.5–2.0 cm in length from 4 wk-old seedlings were cultured on two different basal media (Murashige & Skoog (MS) and Westvaco WV5 (WV5)). All media were supplemented with 6-benzylaminopurine (BAP), to stimulate the formation of axillary buds. Best bud induction was achieved, after 4 wk, on Westvaco WV5 medium (with 10-μM BAP) with rates close to 100%, and an average number of ~ 7 new buds formed per explant. Elongation took place for 6 wk on Westvaco WV5 medium containing activated charcoal (0.2% (w/v)) and without growth regulators. Rooting took place on half-strength Westvaco WV5 medium containing 9.8-μM indole-3-butyric acid (IBA). After 6 wk, root primordia were visible in ~ 43% of shoots. The acclimatization protocol was also optimized by controlling relative humidity, light/photoperiod, temperature, and nutrition, which led to an acclimatization success of ~ 89%. Flow cytometry analysis of DNA-ploidy did not show any variation between micropropagated plants and seedlings. With the protocol here described, it is possible to obtain a high number of genetically uniform plants per explant, 1 yr after in vitro germination of slash pine seeds.An efficient protocol was developed for large-scale micropropagation of slash pine (Pinus elliottii Engelm. var. elliottii). For that, explants consisting of shoot apices 1.5–2.0 cm in length from 4 wk-old seedlings were cultured on two different basal media (Murashige & Skoog (MS) and Westvaco WV5 (WV5)). All media were supplemented with 6-benzylaminopurine (BAP), to stimulate the formation of axillary buds. Best bud induction was achieved, after 4 wk, on Westvaco WV5 medium (with 10-μM BAP) with rates close to 100%, and an average number of ~ 7 new buds formed per explant. Elongation took place for 6 wk on Westvaco WV5 medium containing activated charcoal (0.2% (w/v)) and without growth regulators. Rooting took place on half-strength Westvaco WV5 medium containing 9.8-μM indole-3-butyric acid (IBA). After 6 wk, root primordia were visible in ~ 43% of shoots. The acclimatization protocol was also optimized by controlling relative humidity, light/photoperiod, temperature, and nutrition, which led to an acclimatization success of ~ 89%. Flow cytometry analysis of DNA-ploidy did not show any variation between micropropagated plants and seedlings. With the protocol here described, it is possible to obtain a high number of genetically uniform plants per explant, 1 yr after in vitro germination of slash pine seeds.An efficient protocol was developed for large-scale micropropagation of slash pine (Pinus elliottii Engelm. var. elliottii). For that, explants consisting of shoot apices 1.5–2.0 cm in length from 4 wk-old seedlings were cultured on two different basal media (Murashige & Skoog (MS) and Westvaco WV5 (WV5)). All media were supplemented with 6-benzylaminopurine (BAP), to stimulate the formation of axillary buds. Best bud induction was achieved, after 4 wk, on Westvaco WV5 medium (with 10-μM BAP) with rates close to 100%, and an average number of ~ 7 new buds formed per explant. Elongation took place for 6 wk on Westvaco WV5 medium containing activated charcoal (0.2% (w/v)) and without growth regulators. Rooting took place on half-strength Westvaco WV5 medium containing 9.8-μM indole-3-butyric acid (IBA). After 6 wk, root primordia were visible in ~ 43% of shoots. The acclimatization protocol was also optimized by controlling relative humidity, light/photoperiod, temperature, and nutrition, which led to an acclimatization success of ~ 89%. Flow cytometry analysis of DNA-ploidy did not show any variation between micropropagated plants and seedlings. With the protocol here described, it is possible to obtain a high number of genetically uniform plants per explant, 1 yr after in vitro germination of slash pine seeds.An efficient protocol was developed for large-scale micropropagation of slash pine (Pinus elliottii Engelm. var. elliottii). For that, explants consisting of shoot apices 1.5–2.0 cm in length from 4 wk-old seedlings were cultured on two different basal media (Murashige & Skoog (MS) and Westvaco WV5 (WV5)). All media were supplemented with 6-benzylaminopurine (BAP), to stimulate the formation of axillary buds. Best bud induction was achieved, after 4 wk, on Westvaco WV5 medium (with 10-μM BAP) with rates close to 100%, and an average number of ~ 7 new buds formed per explant. Elongation took place for 6 wk on Westvaco WV5 medium containing activated charcoal (0.2% (w/v)) and without growth regulators. Rooting took place on half-strength Westvaco WV5 medium containing 9.8-μM indole-3-butyric acid (IBA). After 6 wk, root primordia were visible in ~ 43% of shoots. The acclimatization protocol was also optimized by controlling relative humidity, light/photoperiod, temperature, and nutrition, which led to an acclimatization success of ~ 89%. Flow cytometry analysis of DNA-ploidy did not show any variation between micropropagated plants and seedlings. With the protocol here described, it is possible to obtain a high number of genetically uniform plants per explant, 1 yr after in vitro germination of slash pine seeds.An efficient protocol was developed for large-scale micropropagation of slash pine (Pinus elliottii Engelm. var. elliottii). For that, explants consisting of shoot apices 1.5–2.0 cm in length from 4 wk-old seedlings were cultured on two different basal media (Murashige & Skoog (MS) and Westvaco WV5 (WV5)). All media were supplemented with 6-benzylaminopurine (BAP), to stimulate the formation of axillary buds. Best bud induction was achieved, after 4 wk, on Westvaco WV5 medium (with 10-μM BAP) with rates close to 100%, and an average number of ~ 7 new buds formed per explant. Elongation took place for 6 wk on Westvaco WV5 medium containing activated charcoal (0.2% (w/v)) and without growth regulators. Rooting took place on half-strength Westvaco WV5 medium containing 9.8-μM indole-3-butyric acid (IBA). After 6 wk, root primordia were visible in ~ 43% of shoots. The acclimatization protocol was also optimized by controlling relative humidity, light/photoperiod, temperature, and nutrition, which led to an acclimatization success of ~ 89%. Flow cytometry analysis of DNA-ploidy did not show any variation between micropropagated plants and seedlings. With the protocol here described, it is possible to obtain a high number of genetically uniform plants per explant, 1 yr after in vitro germination of slash pine seeds.An efficient protocol was developed for large-scale micropropagation of slash pine (Pinus elliottii Engelm. var. elliottii). For that, explants consisting of shoot apices 1.5–2.0 cm in length from 4 wk-old seedlings were cultured on two different basal media (Murashige & Skoog (MS) and Westvaco WV5 (WV5)). All media were supplemented with 6-benzylaminopurine (BAP), to stimulate the formation of axillary buds. Best bud induction was achieved, after 4 wk, on Westvaco WV5 medium (with 10-μM BAP) with rates close to 100%, and an average number of ~ 7 new buds formed per explant. Elongation took place for 6 wk on Westvaco WV5 medium containing activated charcoal (0.2% (w/v)) and without growth regulators. Rooting took place on half-strength Westvaco WV5 medium containing 9.8-μM indole-3-butyric acid (IBA). After 6 wk, root primordia were visible in ~ 43% of shoots. The acclimatization protocol was also optimized by controlling relative humidity, light/photoperiod, temperature, and nutrition, which led to an acclimatization success of ~ 89%. Flow cytometry analysis of DNA-ploidy did not show any variation between micropropagated plants and seedlings. With the protocol here described, it is possible to obtain a high number of genetically uniform plants per explant, 1 yr after in vitro germination of slash pine seeds.An efficient protocol was developed for large-scale micropropagation of slash pine (Pinus elliottii Engelm. var. elliottii). For that, explants consisting of shoot apices 1.5–2.0 cm in length from 4 wk-old seedlings were cultured on two different basal media (Murashige & Skoog (MS) and Westvaco WV5 (WV5)). All media were supplemented with 6-benzylaminopurine (BAP), to stimulate the formation of axillary buds. Best bud induction was achieved, after 4 wk, on Westvaco WV5 medium (with 10-μM BAP) with rates close to 100%, and an average number of ~ 7 new buds formed per explant. Elongation took place for 6 wk on Westvaco WV5 medium containing activated charcoal (0.2% (w/v)) and without growth regulators. Rooting took place on half-strength Westvaco WV5 medium containing 9.8-μM indole-3-butyric acid (IBA). After 6 wk, root primordia were visible in ~ 43% of shoots. The acclimatization protocol was also optimized by controlling relative humidity, light/photoperiod, temperature, and nutrition, which led to an acclimatization success of ~ 89%. Flow cytometry analysis of DNA-ploidy did not show any variation between micropropagated plants and seedlings. With the protocol here described, it is possible to obtain a high number of genetically uniform plants per explant, 1 yr after in vitro germination of slash pine seeds.An efficient protocol was developed for large-scale micropropagation of slash pine (Pinus elliottii Engelm. var. elliottii). For that, explants consisting of shoot apices 1.5–2.0 cm in length from 4 wk-old seedlings were cultured on two different basal media (Murashige & Skoog (MS) and Westvaco WV5 (WV5)). All media were supplemented with 6-benzylaminopurine (BAP), to stimulate the formation of axillary buds. Best bud induction was achieved, after 4 wk, on Westvaco WV5 medium (with 10-μM BAP) with rates close to 100%, and an average number of ~ 7 new buds formed per explant. Elongation took place for 6 wk on Westvaco WV5 medium containing activated charcoal (0.2% (w/v)) and without growth regulators. Rooting took place on half-strength Westvaco WV5 medium containing 9.8-μM indole-3-butyric acid (IBA). After 6 wk, root primordia were visible in ~ 43% of shoots. The acclimatization protocol was also optimized by controlling relative humidity, light/photoperiod, temperature, and nutrition, which led to an acclimatization success of ~ 89%. Flow cytometry analysis of DNA-ploidy did not show any variation between micropropagated plants and seedlings. With the protocol here described, it is possible to obtain a high number of genetically uniform plants per explant, 1 yr after in vitro germination of slash pine seeds.An efficient protocol was developed for large-scale micropropagation of slash pine (Pinus elliottii Engelm. var. elliottii). For that, explants consisting of shoot apices 1.5–2.0 cm in length from 4 wk-old seedlings were cultured on two different basal media (Murashige & Skoog (MS) and Westvaco WV5 (WV5)). All media were supplemented with 6-benzylaminopurine (BAP), to stimulate the formation of axillary buds. Best bud induction was achieved, after 4 wk, on Westvaco WV5 medium (with 10-μM BAP) with rates close to 100%, and an average number of ~ 7 new buds formed per explant. Elongation took place for 6 wk on Westvaco WV5 medium containing activated charcoal (0.2% (w/v)) and without growth regulators. Rooting took place on half-strength Westvaco WV5 medium containing 9.8-μM indole-3-butyric acid (IBA). After 6 wk, root primordia were visible in ~ 43% of shoots. The acclimatization protocol was also optimized by controlling relative humidity, light/photoperiod, temperature, and nutrition, which led to an acclimatization success of ~ 89%. Flow cytometry analysis of DNA-ploidy did not show any variation between micropropagated plants and seedlings. With the protocol here described, it is possible to obtain a high number of genetically uniform plants per explant, 1 yr after in vitro germination of slash pine seeds. [ABSTRACT FROM AUTHOR]

Embryogenic cultures were obtained from seeds of open-pollinated maritime pine trees representing part of a breeding population. The aim of the present study was to develop and optimize a protocol for cryopreservation of Pinus pinaster somatic embryogenic tissue. The density of the embryogenic suspension and the type of pre-treatment significantly affected the recovery of cryopreserved embryogenic cultures, as evaluated by their survival and regrowth rate. An initial density of the suspension culture of 250 mg/ml improved the regrowth rate of the embryogenic lines. Pre-treatment with maltose 0.4 M significantly increased the regrowth rate when compared to the other tested carbohydrates. Also, the addition of DMSO in a mixture of PEG 4000 and sucrose (PSD solution), instead of DMSO alone, at the same final concentration, was clearly beneficial for recovery of cryopreserved tissues. This improved method for the cryopreservation of P pinaster embryogenic of cultures allowed the successful recovery of 97 percent of the lines stored in liquid of nitrogen.