Development of a regeneration protocol through indirect organogenesis in prickly pear cactus (Opuntia ficus-indica (L.) Mill)

An indirect organogenesis regeneration protocol for Opuntia ficus-indica (L.) Mill var “Blanco sin Espinas” is described. One centimeter square cladode explants sections from previously micropropagated prickly pear plants were cultured in Murashige and Skoog (MS) basal medium supplemented with 20 different combinations of 2,4-dichlorophenoxy acetic acid (2,4-D) and benzyladenine (BA). The best calli induction and regeneration response were observed when 2.26 μM 2,4-D and 2.21 μM BA combination was applied to the nopal explants. Regenerating calli was capable of forming new buds when transferred to MS basal medium supplemented with 0.5 μM BA (proliferation medium). Shoot elongation and rooting were achieved on MS medium without plant growth regulators. Excellent acclimatization to greenhouse conditions was observed for all transferred plantlets. By this procedure no morphological differences were observed between the regenerated and mother plants. This protocol may be also utilized to carry out plant regeneration after genetic transformation, in order to develop transformed plants without the presence of chimeric zones.     

Callus induction and plant regeneration in Cardiospermum halicacabum Linn. an important medicinal plant

Cardiospermum halicacabum Linn. is an important medicinal twining herb belonging to the family sapindaceae. A method for rapid micropropagation of C. helicacabum through plant regeneration from leaf and nodal explant derived calli has been developed. The nodal and leaf segments were cultured on Murashige and Skoog (MS) medium supplemented with 2,4-dichlorophenoxy acetic acid (2,4-D; 0.5–9 μM) for callus induction. Callus production was highest at 5 μM 2,4-D where 96 and 90% of cultured leaf and nodal cuttings produced callus, respectively. The viable calli were maintained at reduced concentration of 2,4-D (2 μM). These calli were transferred to MS medium supplemented with various concentrations of 6-benzyladenine (BA; 2–10 μM) or kinetin (2–10 μM) alone or in combination with indole 3-acetic acid (IAA; 0.2–1.0 μM) for shoot regeneration. The addition of low concentrations of IAA into BA or kinetin containing medium significantly increased the frequency of shoot regeneration in both nodal cuttings and leaf-derived calli. The highest number of adventitious shoots (28 per callus) formed at 8 μM Kin and 0.5 μM IAA. For rooting of the shoots, half-strength MS medium supplemented with different concentrations of indole 3-acetic acid, indole 3-butyric acid (IBA) and (alpha)-naphthalene acetic acid (NAA) 1–5 μM was tried. The optimal result was observed on half-strength MS medium supplemented with 2.5 μM IBA, on which 91% of the regenerated shoots developed roots with an average of 4.2 roots per shoot within 45 days. The in vitro raised plantlets were acclimatized and transferred to soil with 90% success. This in vitro propagation protocol should be useful for conservation as well as mass propagation of this medicinal plant.     

In vitro high frequency direct plant regeneration from whole leaves of blackberry

High frequency and direct (without callus) plant regeneration was achieved from whole leaf explants of thornless blackberry (Rubus hybrid) cv. Black Satin (EC No. 381258; PI No. 553272) in vitro. Leaf blade explants from 1-, 3- and 5-month-old mother cultures were cultured on Murashige and Skoog (MS) medium with thidiazuron (TDZ), N⁶-benzylaminopurine (BAP), indol-3-butyric acid (IBA) and α-naphthalene acetic acid (NAA), alone or in combination. Three-month explants cultured on 0.02 mg l⁻¹ TDZ produced a high regeneration frequency (91.7%) and the most shoots/leaf explant (17.3). The shoot primordia developed within 3 weeks from the point of detachment of the petiole from the leaf blade. The age of the explant source significantly affected the shoot regeneration potential of the leaf explants. Leaves excised from 3-month-old in vitro-cultured shoots performed better than those from 1- and 5-month-old shoots. Shoots rooted best on half-strength MS basal medium with 0.5 mg l⁻¹ IBA and 90% of the plantlets survived acclimatization. The regenerated plantlets were morphologically similar to the mother plants.     

Improvement of caper (Capparis spinosa L.) propagation using in vitro culture and gamma irradiation

Some of the factors influencing the propagation of caper (Capparis spinosa L.) plants in vitro and germination of the seed were studied. The number of adventitious shoots emerging from caper stems cultured in vitro increased from 2.2 shoots per explant when the growth medium contained 2 mg/L of gibberellic acid (GA3) to 5.5 when the growth medium contained 2 mg/L zeatin riboside (ZR) and 1 mg/L naphthalene acetic acid (NAA). The best medium for callus formation from leaf and stem parts contained the growth regulators 1 mg/L 6-benzylaminopurine (BAP) and 0.1 mg/L NAA and the best medium for plant regeneration contained 1 mg/L kinetin and 0.1 mg/L indole-3-acetic acid (IAA). The effect of gamma irradiation on the growth of caper shoots in vitro was also studied. A 10 Gy dose of gamma irradiation stimulated growth of shoots up to 200% and increased shoot rooting percentage from 75 to 100%.     

发根农杆菌介导山定子遗传转化及发根再生植株

为了提高山定子的生根能力,用发根农杆菌转化山定子,诱导产生发根,并由发根诱导出再生植株。通过对不同发根农杆菌株系、培养基、共培养时间以及添加乙酰丁香酮（AS）的研究,优化了发根农杆菌转化体系。当用添加AS（100μmol﹒L-1）的菌液侵染植株切段30 min,共培养3 d,在无植物生长调节剂的固体1/4MS培养基上诱导产生发根时,发根诱导率最高,达到88.89%。将发根根段在MS+BA 2.0 mg﹒L-1 +IBA 0.1 mg﹒L-1+GA3 0.1 mg﹒L-1的再生培养基上,通过愈伤组织的诱导得到再生植株,再生率3%。     

In vitro germination and plantlet establishment of Labisia pumila (Bl.) F. Vill.

In vitro seeds germination and plantlet establishment of Labisia pumila were studied in this report. The seeds obtained from the mature fruits of L. pumila were sterilized and cultured on Murashige and Skoog (MS) solid media supplemented with 1–3 μM of 6-benzylaminopurine (BAP) and 3% (w/v) sucrose. The presence of BAP in the medium significantly affects seeds germination. High percentage of seeds germination (up to 90%) was successfully achieved after 2 weeks of culture on medium supplemented with 2 μM BAP. Up to 70% of explants produced shoots through direct regeneration from newly emerged epicotyls after 5 weeks of culture. The average of 8.1 ± 1.0 shoots per explant obtained on media treated with 2 μM BAP. Seedlings were further transferred to growth media fortified with different types of cytokinin. Result observed after 12 weeks showed that medium supplemented with 1 μM zeatin (ZEA) promote the highest growth with an average of 2.9 ± 1.0 cm shoot length and 7.7 ± 3.2 leaves per explant after 12 weeks. In addition, medium added with 2 μM BAP and supplemented with 3–4% (w/v) of sucrose promote the best growth i.e., 3.0 ± 0.6 shoots per explant, 2.27 ± 0.2 cm length and 4.3 ± 0.5 leaves per explant.     

色素万寿菊叶片再生体系的建立及优化

以色素万寿菊叶片为外植体,探讨了不同的植物生长调节剂组合、AgNO3、凝胶剂及不同的继代培养基对叶片器官发生的影响。结果表明：6-BA 3.0 mg · L-1 + IAA 3.0 mg · L-1 + 蔗糖30.0 g · L-1 + 植物凝胶6.0 g · L-1的MS培养基适宜不定芽的分化,不定芽分化率达97.5%,将分化13 d的叶片外植体转接到组织培养瓶中,30 d后56.7%的不定芽可发育成高约5 cm的植株,再生植株在MS培养基上生根率为100%,移栽成活率达100%。     

In vitro shoot multiplication and plant regeneration in four Capsicum species using thidiazuron

A procedure of in vitro plant propagation using shoot meristem explants (∼0.5 cm) has been developed for Capsicum annuum cv CA960, C. baccatum, C. frutescens and C. praetermissum on Murashige and Skoog [Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Plant Physiol. 15, 473–497] medium containing various cytokinins. Among various concentrations of cytokinins tested; adenine (Ad), N⁶-benzyladenine (BA), kinetin (Kn), zeatin and thidiazuron (TDZ) individually. TDZ regenerated maximum number (4.2–22.4) of shoots in all the Capsicum species tested. Multiple shoot elongation occurred upon transfer to BA (0.22 μM l⁻¹) + IAA (0.48 μM l⁻¹). Rooting of regenerated shoots was achieved on medium supplemented with 5.71 μM l⁻¹ indole-acetic acid (IAA). Rooting was observed in 72–94% of shoots obtained from TDZ-containing regeneration medium followed by elongation treatment in contrast to 8–22% of shoots without elongation treatment. Plantlets obtained from TDZ-containing media were normal diploid (2n = 24) and could readily be established in the soil under green house conditions with a survival frequency of 68–84%. Regenerated plants were developed into morphologically normal, fertile plants and able to set viable seeds.     

Establishment of efficient regeneration protocol from leaf explants of Iris pumila shoots cultured in vitro

An efficient plant regeneration protocol via somatic embyogenesis by leaf base culture of in vitro grown Iris pumila shoots was developed. Induction of embryogenic calli was achieved on MS media supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D), kinetin (4.5 μM, each) and some additives (L-proline, casein hydrolysate, adenine sulphate and tyrosine). Further differentiation of embryogenic calli was achieved on MS hormone-free media, and on media supplemented with either BAP (4.5 μM) or BAP + zeatin (4.5 and 0.2 μM, respectively), which allowed somatic embryos, as well as shoot-like structures to form. Fully developed somatic embryos germinated on an MS hormone-free medium. An anatomical study confirmed that shoot-like structures represented early germinating stages of somatic embryos. Acclimatization of plants derived from somatic embryos was 64% after 1 year and no morphological variation was observed.     

In vitro shoot proliferation and enhancement of rooting for the large-scale propagation of yellow bamboo (Bambusa vulgaris ‘Striata’)

Continuous and rapidly proliferating axillary shoots were raised from axillary buds in secondary branches of adult field culms and nursery grown 1-year-old tissue culture-raised plants of Bambusa vulgaris ‘Striata’. Shoots continuously proliferated in a MS medium containing 4 mg L⁻¹ 6-benzyladenine (BA). The effects of indole butyric acid (IBA) levels, a pretreatment with thidiazuron (TDZ) (1-phenyl-1-([1,2,3-thidiazol-5-yl])urea) and illumination on rooting, were investigated after 6 months of shoot proliferation. A rooting medium with IBA at 3 mg L⁻¹ was optimum for root induction. Shoots of adult field culms that were proliferated in the presence of BA when induced to root in this medium resulted in 40% rooting in 27 days. In vitro shoots raised from 1-year-old tissue cultured plants showed 92% rooting under the same conditions. Rooting was enhanced when the relatively difficult-to-root in vitro shoots from adult field culms were pretreated with 0.5 mg L⁻¹ TDZ for two to three subcultures before placing in the root induction medium. Continuously illuminated shoots pretreated with TDZ for three subcultures showed 100% rooting compared to 83% rooting of shoots that were exposed to a 12 h photoperiod. These findings have been applied in the large-scale propagation of this species.     

Shoot regeneration and genetic transformation by Agrobacterium tumefaciens of Hydrangea macrophylla Ser. leaf discs

A reproducible procedure was developed for genetic transformation of Hydrangea macrophylla Ser. cv. Blaumeise by Agrobacterium tumefaciens following the development of an efficient regeneration system using leaf discs excised from 12 to 15 weeks old meristem-derived vitroplants. Explants were cultivated on solid B5 medium complemented with maltose 110 mM, BAP 10 μM and NAA 0.5 μM. A low light regime of 17 μmol m⁻² s⁻¹ improved regeneration frequency up to 86%. For transformation, leaf discs were inoculated and co-cultivated with two disarmed A. tumefaciens strains, EHA 101 and LBA 4404, both carrying the binary vector pFAJ3000 which contained the nptII selectable gene and the GUS reporter gene. A pre-culture period of 3 days and a short co-cultivation duration (1 day) improved the efficiency of transformation. Inoculation of only 10 min with agitation including (or not) vacuum infiltration was sufficient. If selection on kanamycin containing medium was applied after a 2 weeks culture period on shoot regeneration medium, the percentage of explants forming kanamycin-resistant shoots increased from 3.3 to 13.3%. Integration and expression of the introduced transgene were confirmed by histochemical GUS assay, PCR and Southern blot analysis. Flowering of transgenic plants in glasshouse occurred 10 months after acclimatization.     

Characterization of Prunus domestica L. in vitro regeneration via hypocotyls

Several important aspects of regeneration of European plum from hypocotyl explants were studied. Multiple shoots were induced and full plants were recovered for a large number of plum varieties. This indicates that European plum species is, in general, very responsive to in vitro regeneration from hypocotyls. Shoot organogenesis could be induced from both mature and immature seed explants but regeneration efficiency was higher when immature seeds were used. Rooting efficiency for varieties with low rooting tendency could be greatly increased by addition of naphthaleneacetic acid in the medium. Primary shoots, when sub-cultured on fresh induction medium, produced multiple shoots at a high frequency and such multiplication could continue for many cycles. The secondary new shoots could be induced for various plum varieties. Plant recovery from the secondary shoots was as efficient as that from the primary shoots. This new system may be an alternative for plum transformation with a potential for increasing transformation efficiency. The system can be used for propagation of transgenic lines and other genetic clones of various varieties.     

Plant regeneration of pansy (Viola wittrockiana) ‘Caidie’ via petiole-derived callus

An in vitro plant regeneration protocol for pansy (Viola wittrockiana) cultivar ‘Caidie’ from petioles was established as following: callus induction on a half-strength MS medium supplemented with 0.45 μmol l⁻¹ 2,4-d plus 8.9 μmol l⁻¹ BA, callus subculture on medium F (1/2MS with 4.5 μmol l⁻¹ 2,4-d, 2.7 μmol l⁻¹ NAA and 0.44 μmol l⁻¹ BA) and then on medium T (1/2MS with 4.5 μmol l⁻¹ 2,4-d, 2.7 μmol l⁻¹ NAA and 2.2 μmol l⁻¹ BA), shoot regeneration on medium D3 (MS media supplemented with 2.9 μmol l⁻¹GA₃, 23.6 μmol l⁻¹ AgNO₃, 0.02% active charcoal and 4.5 μmol l⁻¹ TDZ), shoot multiplication on medium M (half-strength MS medium containing NAA 1.1 μmol l⁻¹, TDZ 9.1 μmol l⁻¹ and GA₃ 8.7 μmol l⁻¹), and then shoot elongation and rooting on medium R (MS medium supplemented with 1.1 μmol l⁻¹ NAA and 1.1 μmol l⁻¹ BA). Subculture on appropriate medium was found to be important for successful shoot regeneration.     

In vitro regeneration from mature leaf explants of Pelargonium rapaceum (L.) L’Hérit

Methods to regenerate whole plants from mature leaf explants of Pelargonium rapaceum (L.) L’Hérit were established. To optimize shoot induction, leaf explants were cultured on media containing different types and combinations of plant growth regulators. Growth was initiated within 17–24 days culture, and included callus formation, and root or shoot organogenesis ranging from 20 to 100% regeneration. Shoots were induced only when explants were cultivated on MS medium containing a combination of NAA and kinetin, NAA and BAP, IAA and Kinetin, or IAA and BAP. On media containing NAA and BAP, dark incubation was critical for efficient direct shoot regeneration from explants. Direct shoot formation and the highest number of shoots per explant (17.6) were obtained from leaf explants cultured in the dark for 30 days on MS medium containing 0.1 mg l⁻¹ NAA and 0.1 mg l⁻¹ BAP. Shoots cultured on MS medium containing 0.1 mg l⁻¹ NAA formed tuberous roots with microtubers within 42 days. Healthy regenerated plants were acclimated and transferred to a greenhouse.     

Direct and callus-mediated regeneration of Curcuma soloensis Valeton (Zingiberaceae) and ex vitro performance of regenerated plants

Protocols are outlined for the regeneration of Curcuma soloensis, an attractive tropical ornamental plant, from young vegetative bud explants. We used both direct and callus-mediated regeneration techniques to produce material suitable for mass propagation and the development of transgenic plants. During direct plantlet propagation, the presence of thidiazuron (TDZ) in the growing medium induced more than three times as many shoots as 6-benzylaminopurine (BA), with a mean of 18.7 shoots per explant on MS medium containing 2.5 μM TDZ compared to 5.0 shoots with 40 μM BA. Subsequently, the shoots rooted readily on MS basal medium that was free of plant growth regulators. During indirect plantlet regeneration, TDZ combined with BA and 2,4-dichlorophenoxyacetic acid (2,4-D) had significant effects on embryogenic callus induction and multiplication. The frequency of callus formation was 91.1% for explants cultured on MS basal medium supplemented with 2.5 μM TDZ, 2.0 μM BA and 1.2 μM 2,4-D. On average 7.1 shoots were produced per callus mass cultured on MS medium supplemented with 2.5 μM TDZ, 9.0 μM BA and 1.2 μM naphthaleneacetic acid (NAA). Regenerated shoots were transferred to MS medium supplemented with 2.5 μM TDZ, to produce multiple shoots. In vitro cultured plantlets readily acclimatized to greenhouse conditions, showing 100% survival rates in a sphagnum, perlite and sand (1:1:1) medium. These plants were transplanted into pots or planted in the field. The ex vitro acclimated plants grew vigorously and produced showy inflorescences 5–6 months after planting. The high-frequency of shoot multiplication and rapid flowering of tissue-cultured plants indicate that C. soloensis has great potential in the floricultural market.     

Somatic embryogenesis and plant regeneration in Psidium guajava L. cv. Banarasi local

A protocol for plant regeneration by somatic embryogenesis was developed in guava cv. Banarasi local by using immature zygotic embryo explants. Best induction of somatic embryogenesis was achieved from 10-week-old zygotic embryos on MS medium supplemented with 2,4-d (4.52 μM) and 5% sucrose. Maximum number of somatic embryos was produced when zygotic embryo explants were transferred to growth regulator free full strength MS basal medium after 8 days treatment with 2,4-d. Full strength MS basal medium containing 5% sucrose was most favorable for maturation of somatic embryos. Highest frequency of conversion and normal plantlet production were recorded from elongated torpedo stages of somatic embryos on half strength MS medium containing 3% sucrose. Over 90% of rooted shoots survived acclimatization.     

Root induction in three species of bamboo with different rooting abilities

Rooting of in vitro axillary shoots from adult field culms of Bambusa atra, Dendrocalamus giganteus and D. hookeri and of juvenile seedling shoots of D. giganteus were investigated. B. atra rooted spontaneously without exogenous auxin during axillary shoot proliferation, while both Dendrocalamus species rooted only on transfer to rooting media with indole-3-butyric acid (IBA). D. giganteus required coumarin, an auxin protector, in addition to IBA. Rooting in adult shoots of D. giganteus was lower (45.6%) than that in the juvenile shoots (96.7%) but adult shoots of D. hookeri rooted well (88.9%). A pretreatment with thidiazuron (TDZ: N-phenyl-N-[(1,2,3-thidiazol-5-yl)urea]) induced development of axillary buds and subsequently 95% rooting in adult D. giganteus shoots but had no significant effect on rooting in juvenile shoots of D. giganteus or D. hookeri. Pretreatment with tri iodobenzoic acid (TIBA) arrested shoot and bud development leading to very low or no rooting in all three species.     

Effect of gibberellic acid (GA3) on elongation and rooting of ‘St. Julien A’ rootstock in vitro

‘St. Julien A’ (Prunus instititia L.) rootstock was induced to proliferate shoots on a modified half-strength Murashige and Skoog (MS) medium. Cultures treated with 12.5 mg l^?1 gibberellic acid (GA₃) produced elongated shoots suitable for rooting. Elongated shoots were placed in media with indolebutyric acid (IBA) or indole-3-acetic acid (IAA) with or without a 16-day dark incubation. Light (16-h photoperiod) inhibited rooting. IAA (4 mg l^?1) was ineffective in promoting rooting. Rooting was best when shoots were incubated in the dark with IBA (4 mg l^?1). GA₃ was deleterious to shoots, causing chlorosis and apical die-back. Light regime interacted with auxin treatments in affecting shoot condition. Shoot condition was better on shoots treated with IBA and dark-incubated; while those treated with IAA were better when light-incubated.     

In vitro propagation of Lychnis senno Siebold et Zucc., a rare plant with potential ornamental value

Lychnis senno is a rare and valued ornamental plant. Seed propagation is not efficient because of the low germination rate. To grow commercially L. senno in China, a protocol for in vitro germination and propagation of this species was developed. Various germination rates were obtained by treating seeds with GA₃ during 1–6 months storage period. The highest germination rate reached 19.4% when seeds were treated with 250 mg/l GA₃ and stored for 5 months at 4 °C. Axillary shoot proliferation was induced in the nodal segments of the seedlings on medium containing specific concentrations of BA and NAA [Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol. Plant 15, 473–497]. Maximum number of shoots was developed on a medium supplemented with 5 mg/l BA and 0.5 mg/l NAA, while the higher shoots were observed on a medium supplemented with 0.5 mg/l BA and 0.05 mg/l NAA. Rooting was induced in 91.7% of the regenerated explants on a half-strength MS medium supplemented with 0.5 mg/l NAA. The plantlets grew well and flowered after transfer to the greenhouse. The chromosome numbers of seedlings and propagated plants were also determined to be 2n = 2x = 24.     

High frequency in vitro embryogenic callus induction and plant regeneration from indiangrass mature caryposis

Indiangrass [Sorghastrum nutans (L.) Nash.] is native to the North America and is an important component of the original tall grass prairie. It is also an important ornamental and forage grass. Recently, it has been proposed as an ideal biomass producer for cellulosic ethanol production. Genetic transformation is an important tool for introducing important agronomic traits into plants, but an efficient and reproducible in vitro regeneration protocol is a prerequisite for successful genetic transformation. In this report, we used mature caryopses as explants and tested the effect of various combinations of 2,4-dichlorophenoxyacetic acid (2,4-D) (1–5) and kinetin (KT) (0, 0.1, and 0.2) on embryogenic callus induction using LS basal medium. Caryopses cultured on media supplemented with 2,4-D alone generally outperformed those cultured on media supplemented with both 2,4-D and kinetin for embryogenic callus induction. The best treatment is LS basal medium supplemented with 3 mg l⁻¹ 2,4-D. LS basal medium supplemented with KT of 0, 0.5, 1, 2 or 5 mg l⁻¹ were tested for regeneration efficiency which was shown to increase as the KT concentration increased. The quality of the shoots produced on the medium containing KT at 5 mg l⁻¹, which produced the highest regeneration frequency appeared to be lower as leaves become vitrified. Shoots were moved to a rooting medium containing either 0 or 0.1 mg l⁻¹ α-naphthaleneacetic acid (NAA). Rooted plantlets were then transferred to soil-containing pots and were placed in a mist room for 1 week before they are transferred to a normal greenhouse where they all survived. The reported regeneration protocol is very efficient and highly reproducible in spite of the heterogeneous nature of the tested cultivar; thus it should be suitable for genetic transformation.