<Emphasis Type="Italic">In vitro</Emphasis> adventitious shoot bud differentiation and plantlet regeneration in <Emphasis Type="Italic">Feronia limonia</Emphasis> L. (Swingle)

Summary In vitro adventitious shoot bud regeneration systems are considered most suitable for Agrobacterium-and biolisticsmediated genetic transformation to obtain transgenic plants. In the present investigation, multiple adventitious shoot buds could be induced directly from Feronia limonia hypocotyl explants inoculated on Murashige and Skoog (MS) medium containing different growth regulators. During the initial phase, the hypocotyl segments nearer to the cotyledons responded quickly compared to those closer to the root. The response, however, was comparable in both the segments in subsequent subculture. Of the various cytokinins, 2.22 μM 6-benzylaminopurine (BA) proved to be more effective compared to kinetin (Kn). The two-way interaction of BA and Kn significantly influenced shoot regeneration and contributed the most among the interactions studied. The best response, however, was obtained when 2.22 μM BA and 2.32 μM Kn were combined. Although the effect of auxins like α-naphthaleneactic acid (NAA) combined with cytokinins evoked a significant responsein terns of number of shoot buds, this response did not supersede the effect of combined cytokinins. Vone of the polyamines tested induced shoot buds on hypocotyl segments. Adventitious shoots were multiplied on MS medium containing 2.22 μM BA, 6.96 μM Kn, and 0.05 μM NAA. More than 60% of the shoots produced roots when cultured on medium containing one-quarter strength MS salts, 10% suerose, 0.6% agar, and 7.36μM indole-3-butyric acid. The adventious origin of shoot buds showing continuous vascular connections was confirmed through histological investigations.     

Axillary bud proliferation and organogenesis of <Emphasis Type="Italic">Euphorbia pulchurrima</Emphasis> winter rose

Summary Protocols for both axillary bud proliferation and shoot organogenesis of Euphorbia pulchurrima Winter Rose^TM were developed using terminal buds and leaf tissues. Greenhouse-grown terminal buds were placed on Murashige-Skoog (MS) basal medium supplemented with various concentrations of either benzlyaminopurine (BA) or thidiazuron (TDZ). Explants produced the greatest number of axillary buds on media containing between 2.2 and 8.8 μM BA. The number of explants that produced axillary buds increased with increasing BA concentration. TDZ at concentrations between 2.3 and 23.0 μM caused hyperhydricity of shoots and were not effective in promoting shoot proliferation. The most calluses and shoots were produced from leaf midvein sections from in vitro grown plants placed on the medium containing 8.8–13.3 μM BA and 17.1 μM indole-3-acetic acid (IAA) for 1 mo. before transferring to the medium containing only BA. Adventitious buds were produced only from red-pigmented callus, and explants that produced callus continued to produce adventitious shoots in the presence of IAA. Five-mo.-old shoots derived from shoot culture or organogenesis rooted readily in artificial soil with or without treatment with indolebutyric acid, and were acclimatized in the greenhouse.     

Micropropagation of <Emphasis Type="Italic">Scabiosa caucasica</Emphasis> Bieb. Cv. Caucasica blue

Summary Micropropagation of Scabiosa caucasica cv. Caucasica Blue was achieved by culturing, separating axillary and adventitious shoots, or node sectioning on Murashige and Skoog (MS) medium supplemented with benzyladenine (BA). The highest frequency of adventitious shoots regenerated from nodal or internodal explants and leaf blade (with or without petiole) appeared to occur on MS medium with 4.4 and 18 μM BA, respectively. Addition of 0.19 or 1.9 μM α-naphthaleneacetic acid to the BA-containing medium promoted callus formation and reduced shoot organogenesis. During micropropagation, shoot nodal explants derived from in vitro shoots cultured on MS medium supplemented with 4.4 μM BA yielded 8.9 shoots per explant within 40 d after culture initiation.     

<Emphasis Type="Italic">In vitro</Emphasis> bud regeneration of <Emphasis Type="Italic">Carthamus tinctorius</Emphasis> and wild <Emphasis Type="Italic">Carthamus</Emphasis> species from leaf explants and axillary buds

The organogenic competence of leaf explants of eleven Carthamus species including C. tinctorius on Murashige and Skoog (MS) medium supplemented with different concentrations of thidiazuron (TDZ) + α-naphthaleneacetic acid (NAA) and 6-benzyladenine (BA) + NAA was investigated. Highly prolific adventitious shoot regeneration was observed in C. tinctorius and C. arborescens on both growth regulator combinations and the shoot regeneration frequency was higher on medium supplemented with TDZ + NAA. Nodal culture of nine Carthamus species on media supplemented with BA and kinetin (KIN) individually revealed the superiority of media supplemented with BA over that of KIN in facilitating a higher shoot proliferation index. Proliferating shoots from axillary buds and leaf explants were transferred to medium supplemented with 1.0 mg dm⁻³ KIN or 0.5 mg dm⁻³ BA for shoot elongation. Elongated shoots were rooted on half-strength MS medium supplemented with 1.0 mg dm⁻³ each of indole-butyric acid (IBA) and phloroglucinol. The plantlets thus obtained were hardened and transferred to soil.     

Plant regeneration via organogenesis from adventitious bud explants of a medicinal herb species, <Emphasis Type="Italic">Polygonatum cyrtonema</Emphasis>

Summary Explants derived from adventitious buds, rhizomes, stems, and leaves of a medicinal plant, Polygonatum cyrtonema, were studied for plantlet regeneration, and only adventitious bud explants were able to be regenerated into plantlets. Regeneration was also accompanied by the formation of rhizome-like tissue, the medicinal portion of the plant. The optimum hormone combination for plantlet regenertion was 4.44 μM benzyladenine plus 2.26 μM 2,4-dichlorophenoxyacetic acid, at which new adventitious buds were obtained from 96.6% of the adventitious bud explants, with an average of 5.2 buds per explant. The best medium for root induction was half-strength Murashige and Skoog medium with 4.57 μM α-naphthaleneacetic acid, as 92% of regenerated buds rooted. Regenerated plantlets were successfully transferred to a greenhouse with 86% survival. Histological observation indicated that new adventitious buds originated from the superficial meristematic cell cluster of the granular callus induced from adventitious bud explants via organogenesis.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration of the tennessee coneflower (<Emphasis Type="Italic">Echinacea tennesseensis</Emphasis>)

Summary Tennessee coneflower [Echinacea tennesseensis (Beadle) Small] was regenerated from flower stalks, leaf sections from flowering plants, and hypocotyls and cotyledons from seedlings. Murashige and Skoog medium (MS) supplemented with naphthaleneacetic acid (NAA) at 0.54 μM and thidiazuron (TDZ) at 22.7 μM yielded the most shoots per leaf explant. NAA and 6-benzylaminopurine concentrations for optimal shoot regeneration from leaf, flower stalk, cotyledon and hypocotyl explants in MS media were 0.54 and 24.6μM, respectively. All explant types generated shoots; however, those derived from leaves and flower stalks produced the highest number of shoots per explant and highest percentage of explants with shoots. Explants cultured on media containing high levels of NAA (5.4–27 μM) formed calluses but no adventitious shoot. Leaf explants responded to a wider range of NAA concentrations than the other explant types but shoots generated from flower stalks grew the fastest. While all cytokinins tested increased the number of shoots per explant, the number of shoots in media containing TDZ was increased by nearly threefold. Regenerated shoots from all explant types cultured on MS medium supplemented with 0.25 μM indole-3-butyric acid initiated roots within 4 wk; NAA was not effective for root induction. All vernalized plantlets developed into plants that were morphologically identical to the source material.     

Direct shoot regeneration from leaf explants of <Emphasis Type="Italic">Rosa damascena</Emphasis> Mill.

Summary A protocol for in vitro propagation using direct induction of shoot buds from leaf explants of in vitro-raised shoots of Rosa damascena var. Jwala is reported. The present study is the first report on direct shoot regeneration in scented roses. Elite plants raised from nodal explants and maintained for over 2yr in vitro on a static liquid shoot multiplication Murashige and Skoog (MS) medium supplemented with 5.0 μM benzyladenine (BA) and 3% sucrose were used. Petioles from fully developed young leaves, obtained after 4 wk of pruning of old shoots, were found to be ideal for regeneration of shoots. Initially the explants were cultured in an induction medium [half-strength MS+3% sucrose+6.8μM thidiazuron+0.27 μM α-naphthaleneacetic acid (NAA)+17.7 μM AgNO₃] and subsequently transferred to the regeneration medium (MS+2.25 μM BA+0.054 μM NAA) after 7, 14, 21, 28, and 35d. The highest shoot regeneration response (69%) was recorded when shoots were kept in the induction medium for 21 d and later transferred to regeneration medium. Histological studies revealed direct formation of shoot buds without the intervening callus phase. In vitro rooting of micro-shoots was accomplished within 2wk on half-strength MS liquid medium supplemented with 10.0 μM IBA and 3% sucrose for 1 wk in the dark and later transferred to hormone-free medium and kept in the light. Plantlets, remaining in the latter medium for 5–6 wk when transferred to soil, showed 90% survival.     

Adventitious bud regeneration from leaf and cotyledon explants of Chinese hawthorn (<Emphasis Type="Italic">Crataegus pinnatifida</Emphasis> Bge. var. <Emphasis Type="Italic">major</Emphasis> N.E.Br.)

Hawthorn (Crataegus spp.) is an important plant with a long history as an ornamental and a source of medicine. A protocol is outlined for adventitious bud regeneration from leaf and cotyledon explants of Chinese hawthorn (C. pinnatifida Bge. var. major N.E.Br.). Adventitious buds were induced on both the leaves of sprouting winter buds and the leaves of in vitro plants, but the percentage of bud regeneration from leaves of in vitro plants was very low—less than 6%. On N6 medium supplemented with 31.08 μM BA and 9.67 μM NAA, the percentages of bud regeneration from leaves of sprouting winter buds of cultivars “Liaohong” and “Qiujinxing” were 31.4% and 17.6%, respectively. The regeneration abilities of three kinds of cotyledon explants, immature cotyledon, mature cotyledon, and cotyledon leaf, were compared. The percentage of bud regeneration from cotyledon leaves was higher. On MS media supplemented with 4.44 μM BA and 4.54–9.08 μM TDZ, the percentages of bud regeneration from cotyledon leaves of cultivars “Qiujinxing” and “Xiajinxing” were 27.7 ± 7.8% and 20.1 ± 4.7%, respectively, and the numbers of buds per explant were 5.9 ± 1.6 and 3.2 ± 0.7, respectively. On B5 medium supplemented with 2.22 μM BA, 2.32 μM Kn, and 0.57 μM IAA, adventitious buds grew quickly and 80–100% of buds developed into shoots. The shoots rooted successfully with the two-step rooting method. Ninety days after transplantation, more than 80% plants were survived. This system of adventitious bud regeneration from leaf and cotyledon explants could be useful for the genetic transformation and polyploidization of Chinese hawthorn.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration of <Emphasis Type="Italic">Salvia nemorosa</Emphasis> L. from shoot tips and leaf explants

Summary Shoot tips and leaves excised from in vitro shoot cultures of Salvia nemorosa were evaluated for their organogenic capacity under in vitro conditions. The best shoot proliferation from shoot tips was obtained on Murashige and Skoog (MS) medium supplemented with 8.9 μM 6-benzylaminopurine (BA) and 2.9 μM indole-3-acetic acid (IAA). Leaf lamina and petiole explants formed shoots through organogenesis via callus stage and/or directly from explant tissue. The highest values for shoot regeneration were obtained with 0.9 μM BA and 2.9 μM IAA for lamina explants. No shoot organogenesis was obtained on leaf explants cultured on MS medium supplemented with α-naphthaleneacetic acid (NAA). The regenerated shoots rooted the best on MS medium containing 0.6 μM IAA or 0.5 μM NAA. In vitro-propagated plants were transferred to soil with a survival rate of 85% after 3 mo.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of <Emphasis Type="Italic">Decalepis arayalpathra</Emphasis>, a critically endangered ethnomedicinal plant

Summary This study reports a protocol for successful micropropagation of Decalepis arayalpathra (Joseph and Chandras) Venter. (Janakia arayalpathra Joseph and Chandrasekhran; Periplocaceae), a critically endangered and endemic ethnomedicinal plant in the southern forests of the Western Ghats which is overexploited for its tuberous medicinal roots by the local Kani tribes. Natural regeneration is rare and conventional propagation is difficult. Conservation of the species through micropropagation was attempted. The nodal explants of greenhouse-raised plants, were more desirable than cotyledonary nodal explants of aseptic seedlings. The basal nodes (73%) of 12–16-wk-old greenhouse-grown plants cultured in Murashige and Skoog (MS) medium containing 12.96 μM 6-benzyladenine (BA), 2.48 μM 2-isopentenyladenine (2-ip) and 2.68 μM α-naphthaleneacetic acid (NAA) formed 16–17 cm long unbranched robust solitary shoots in 8 wk. Cotyledonary nodal explants cultured in the same medium showed multiple shoot formation and axillary branching. But the shoots were thin, fragile and not suitable for mass propagation. Single nodes of a solitary shoot subcultured on MS medium containing 2.22 μM BA and 0.24 μM 2-ip together produced 9.8±0.3 nodes from 18.0±0.6 cm long shoots within 5–6 wk. The basal nodes of the shoots so formed were repeatedly subcultured to increase the stock of propagules while the 2.5–3.0 cm terminal cuttings were used for rooting. The best root induction (68%) and survival (86%) was achieved on half-strength MS medium supplemented with 1.07 μM NAA. Field-established plants showed uniform growth and phenotypic similarity to parental stock.     

<Emphasis Type="Italic">In vitro</Emphasis> shoot regeneration from cotyledonary node explants of a multipurpose leguminous tree <Emphasis Type="Italic">Pterocarpus marsupium</Emphasis> Roxb.

Summary A protocol has been developed for in vitro plant regeneration from cotyledonary nodes of Pterocarpus marsupium Roxb. Multiple shoots were induced from cotyledonary nodes derived from 20-d-old axenic seedlings grown on Murashige and Skoog (MS) medium containing 2.22–13.32 μM benzyladenine (BA) or 2.32–13.93 μM kinetin alone or in combination with 0.26 μM α-naphthaleneacetic acid (NAA). The highest frequency of responding explants (85%) and maximum number of shoots per explant (9.5) were obtained on MS medium supplemented with 4.44 μM BA and 0.26 μM NAA after 15 wk of culture. A proliferating shoot culture was established by repeatedly subculturing the orginal cotyledonary nodal explant on fresh medium after each harvest of the newly formed shoots. Nearly 30% of the shoots formed roots after being transferred to half-strength MS medium containing 9.84 μM indole-3-butyric acid after 25 d of culture. Fifty percent of shoots were also directly rooted as microcuttings on peat moss, soil, and compost mixture (1∶1∶1). About 52% plantlets rooted under ex vitro conditions were successfully acclimatized and established in pots.     

<Emphasis Type="Italic">In vitro</Emphasis> culture studies on <Emphasis Type="Italic">Stevia rebaudiana</Emphasis>

Summary Shoot apex, nodal, and leaf explants of Stevia rebaudiana Bertoni can regenerate shoots when cultured on Murashige and Skoog (MS) medium supplemented with 6-benzyladenine (BA; 8.87 μM) and indole-3-acetic acid (5.71 μM). Rooting of the in vitro-derived shoots could be achieved following subculture onto auxin-containing medium. A survival rate of 70% was recorded at the hardening phase on the substrate cocopeat. The presence of the sweet diterpene glycosides, viz. stevioside and rebaudioside, was confirmed in the in vitro-derived tissues of Stevia using HPTLC techniques. Callus cultured on agar-solidified MS medium supplemented with BA (8.87 μM) and indole-3-butyric acid (9.80 μM) showed the highest sweetener content.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of an endangered plant species, <Emphasis Type="Italic">Thermopsis turcica</Emphasis> (Fabaceae)

This report deals with micropropagation of the critically endangered and endemic Turkish shrub, Thermopsis turcica using callus, root and cotyledonary explants. Callus cultures were initiated from root and cotyledon explants on MS medium supplemented with 0.5–20 μM NAA or 2,4-D. The root explants were found to be better in terms of quick responding and callusing percentages as compared to the cotyledons. Organogenic callus production with adventitious roots and shoots were obtained on MS medium with only NAA. The calli obtained with NAA, root and cotyledonary explants were cultured with BA and kinetin (2–8 μM) alone or in combination with a low level (0.5 μM) of 2,4-D or NAA. The best regeneration of shoots from root explants was observed on hormone-free MS medium. NAA with BA or kinetin in the medium improved shoot induction from the calli obtained with NAA. Maximum percentage of shoots (93.3%), maximum number of shoots (6.2) and maximun length of shoots (8.22 cm) were achieved from cotyledonary explants at 4 μM BA and 0.5 μM NAA. The presence of 0.5 μM or higher levels of 2,4-D in shoot induction medium inhibited the regeneration in T. turcica explants. 83% of in vitro rooting was attained on pulsed-IBA treated shoots. The regenerated plants with well developed shoots and roots were successfully acclimatized. Application of this study’s results has the potential to conserve T. turcica from extinction.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of <Emphasis Type="Italic">Zingiber petiolatum</Emphasis> (Holttum)

Summary We describe an in vitro propagation protocol for Zingiber petiolatum (Holttum), I. Theilade, a rare species from the southern part of Thailand. Fruits were surface-sterilized and seeds germinated on Murashige and Skoog medium (MS) medium supplemented with 3% sucrose. Three-month-old seedlings were used as initial plant material for in vitro propagation. Terminal buds of the plants were inoculated on MS medium containing 6-benzylaminopurine (BA; 2.2–35.5 μM) alone or in combination with 1-naphthaleneacetic acid (0.5 μM). Eight weeks after inoculation, the cultures were transferred to MS medium without plant growth regulators for 4wk. The cultures transferred from MS medium with 17.8 μM BA revealed the highest shoot induction rate of 6.1±0.7 shoots per explant. Rooting was spontaneously achieved in MS medium without plant growth regulators. Rooted plants were successfully transplanted to soil.     

Influence of auxins in direct <Emphasis Type="Italic">in vitro</Emphasis> morphogenesis of <Emphasis Type="Italic">Euphorbia nivulia</Emphasis>, a lectinaceous medicinal plant

Summary Somatic embryo (bipolar) or shoot (monopolar) morphogenesis in mesophyll cells of Euphorbia nivulia Buch.-Ham in vitro was dependent on the type of auxin supplementing Murashige and Skoog (MS) medium containing benzyladenine. Direct in vitro morphogenesis, i.e., organogenesis, and somatic embryogenesis were significantly influenced by seasonal growth of the donor plant, explant position (proximal, mid, and distal), and light. Explants collected in march/April were superior to July/August material. Proximal explants underwent morphogenesis more readily than mid- and tip-derived explants. Incubation in the light favored morphogenesis while darkness was inhibitory. Kinetin (Kn) was also inhibitory to morphogenesis. MS medium enriched with different levels of N⁶-benzyladenine (BA) alone, or in combination with α-naphthaleneacetic acid (NAA) or indole-3-acetic acid (IAA), induced adventitious shoots directly. Explants collected in March/April cultured on medium with 13.3 μM BA and 2.69 μM NAA developed the highest number of shoots, a mean of 15.2 shoots per proximal explant. Developed shoots rooted the best on half-strength MS medium with 2.46 μM indole-3-butyric acid, which developed a mean of 5.2 roots per shoot. Rooted healthy shoots could be transplanted to small pots, with an 80% survival rate. Addition of 2,4-dichlorophenoxyacetic acid (2.4-D) to BA-supplemented medium was obligatory to develop somatic embryos. MS medium containing 2.26 μM 2,4-D and 4.44 μM BA induced a mean of 44.8 somatic embryos per proximal explant. The embryos passed through distinct stages of embryogenesis, namely globular, heart, torpedo, and early cotyledonary. The embryos (88%) underwent maturation on half-strength MS medium with 2.89 μM gibberellic acid (GA₃), and its subsequent transfer on half-strength MS basal medium in light conditions facilitated 80% conversion of embryos to plantlets. Direct shoots or embryos were originated from the mesophyll cells. Somatic embryo development was concurrent with the independent origin of vasculature in the bulbous basal portion. The survival rate of embryo-derived plants was 90%.     

Effect of auxins on indirect <Emphasis Type="Italic">in vitro</Emphasis> morphogenesis and expression of <Emphasis Type="Italic">gusA</Emphasis> transgene in a lectinaceous medicinal plant, <Emphasis Type="Italic">Euphorbia nivulia</Emphasis> Buch.-Ham.

Summary The types of auxin in Murashige and Skoog (MS) medium containing N ⁶-benzyladenine (BA) determined indirect morphogenesis, i.e. development to bipolar somatic embryos or monopolar shoots in Euphorbia nivulia Buch.-Ham. Indirect in vitro morphogenesis depended on growth regulators, explant excision period, and light. Calli induced from explants collected in March–April were superior in the induction of indirect morphogenesis to those collected in July–August. Light enforced in vitro morphogenesis, while darkness was inhibitory. The presence of kinetin in the medium also inhibited morphogenesis. Calli developed on explants collected in March–April grown on MS medium fortified with α-naphthaleneacetic acid (NAA) and BA facilitated indirect organogenesis, while those developed on medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) and BA underwent somatic embryogenesis. MS medium with 13.3 μM BA and 2.69 μM NAA was the best for induction of shoots from callus, which developed a mean of 15.7 shoots. Shoots were best rooted on half-strength MS medium enriched with 2.46 μM indole-3-butyric acid with a mean of 5.1 roots per shoot. MS medium supplemented with 2.26 μM 2,4-D and 4.44 μM BA induced the highest number (mean of 13.4) of somatic embryos. Of the embryos transferred on half-strength MS medium containing 2.89 μM gibberellic acid, 78% of embryos developed to the cotyledonary stage. Most cotyledonary embryos (80%) underwent conversion to plantlets upon being transferred to half-strength MS basal medium in light. The survival rate of organogenesis and embryo-derived plants was 80 and 90%, respectively. Calli transformed with Agrobacterium tumefaciens showed expression of the gusA transgene and resistance to kanamycin, but did not undergo morphogenesis.     

High-frequency plant regeneration from cotyledon callus of <Emphasis Type="Italic">Acacia sinuata</Emphasis> (Lour.) Merr

Summary A new reliable protocol for the induction of adventitious shoots and plant regenertion from cotyledon-derived callus of Acacia sinuata has been developed. Calluses were induced from cotyledon explants on Murashige and Skoog (MS) medium containing 3% sucrose, 0.8% agar or 0.15% phytagel, 8.1 μM α-naphthaleneacetic acid, and 2.2 μM 6-benzylaminopurine (BA). High-frequency regeneration of adventitious buds from callus was achieved when cultured on half-strength MS medium supplemented with 10% coconut water, 13.3 μM BA, and 2.5 μM zeatin. Histological studies revealed that the regenerated shoots originated from the callus. Among the various carbohydrates tested, sucrose at 87.6 mM was optimum for shoot-bud induction. Addition of 1.7 μM gibberellic acid along with 4.4 μM favored shoot elongation. In vitro-raised shoots produced prominent roots when transferred to half-strength MS medium supplemented with 7.4 μM indole-3-butyric acid. Rooted plants, thus developed, were hardened and successfully established in soil (45%). This protocol yielded an average of 40 plantlets per cotyledon explant over a period of 3 mo.     

Plant regeneration by somatic embryogenesis and organogenesis in commercial pineapple (<Emphasis Type="Italic">Ananas comosus</Emphasis> L.)

Summary Axillary and terminal buds from suckers of Ananas comosus cv. Phuket were established on Murashige and Tucker-based (MT) medium with 2.0 mgl⁻¹ (9.8 μM) indolebutyric acid, 2.0 mgl⁻¹ (10.74 μM) naphthaleneacetic acid, and 2.0 mgl⁻¹ (9.29 μM) kinetin, followed by multiplication on Murashige and Skoog-based (MS) medium containing 2.0 mgl⁻¹ (8.87 μM) benzyladenine (BA) to provide a continuous supply of axenic shoots. Leaves, excised from such cultured shoots, produced adventitious shoots from their bases when these explants were cultured on MS medium containing 0.5 mgl⁻¹ (2.26 μM) 2,4-dichlorophenoxyacetic acid (2,4-D) and 2.0 mgl⁻¹ (8.87 μM) BA. Embryogenic callus was produced when leaf explants were cultured on MS medium with 3.0 mgl⁻¹ (12.42 μM) 4-amino-3,5,6-trichloropicolinic acid (picloram). Somatic embryos developed into shoots following transfer of embryogenic tissues to MS medium with 1.0 mgl⁻¹ (4.44 μM) BA. Cell suspensions, initiated by transfer of embryogenic callus to liquid MS medium with 1.0 mgl⁻¹ (4.14 μM) picloram or 1.0 mgl⁻¹ (4.52 μM) 2,4-D, also regenerated shoots by somatic embryogenesis, on transfer of cells to semisolid MS medium with 1.0 mgl⁻¹ (4.44 μM) BA. All regenerated shoots rooted on growth regulator-free MS medium, prior to ex vitro acclimation and transfer to the glasshouse. These studies provide a baseline for propagation, conservation, and genetic manipulation of elite pineapple germplasms.     

In vitro morphogenesis of organogenic nodules derived from <Emphasis Type="Italic">Sclerocarya birrea</Emphasis> subsp. <Emphasis Type="Italic">caffra</Emphasis> leaf explants

Sclerocarya birrea (marula) is an indigenous South African tree with highly valued medicinal and nutritional properties. Induction of nodular meristemoids from leaf explants was achieved on Murashige and Skoog (MS) and woody plant medium (WPM) supplemented with 6-benzyladenine (BA) in combination with naphthalene acetic acid (NAA), indole-3-butryric acid (IBA) and indole-3-acetic acid (IAA). Induction of nodular meristemoids from 86% of the leaf cultures was achieved on MS medium with 4.0 μM BA and 1.0 μM NAA. High levels (78–100%) of induction were also achieved on WPM with different concentrations of BA (1.0–4.0 μM) and IBA (1.0–4.0 μM). The highest conversion of meristemoids into shoots was only 22% for 4.0 μM BA and 1.0 μM NAA on MS initiation medium. This was improved to 62% when nodular clusters were cultured in a MS liquid medium. Histological studies revealed the globular stage of the nodular meristemoids. This protocol has potential for application in mass micropropagation and plant breeding of S. birrea.     

Tissue culture and plant regeneration of the salt marsh monocots <Emphasis Type="Italic">Juncus roemerianus</Emphasis> and <Emphasis Type="Italic">Juncus gerardi</Emphasis>

Summary Tissue culture and plant regeneration protocols for the salt marsh plants Juncus roemerianus Scheele and Juncus gerardi Loisel, were developed. J. roemerianus callus was induced from mature seeds cultured on Murashige and Skoog (MS) medium supplemented with 2.22 μM 6-benzylaminopurine (BA), 5.37 μM α-naphthaleneacetic acid (NAA), 2.26 μM 2,4-dichlorophenoxyacetic acid (2,4-D), and 50 ml l⁻¹ coconut water (callus induction medium). The callus was subcultured on MS medium containing 2.22 μM BA, 5.37 μM NAA, and 9.05 μM 2,4-D for callus maintenance. Shoot regeneration occurred 2 wk after transferring the callus onto shoot regeneration medium, which consisted of MS medium containing BA or thidiazuron. A high frequency of shoot regeneration was obtained when the medium contained 13.3 μM BA. Regenerated shoots were transferred to MS medium supplemented with 10.7 μM NAA for root production. Rooting did not occur in the shoots regenerated on the thidiazuron-containing media. The callus induction medium for J. roemerianus was also effective in inducing callus of J. gerardi from young inflorescences. The same medium was also used for callus maintenance. Shoot regeneration occurred 10 d after transferring the callus onto MS medium supplemented with 0.44 μM BA and 0.57 μM indole-3-acetic acid. Root regeneration occurred after transferring the shoots onto MS medium plus 0.44 μM BA and 14.8 μM indole-3-butyric acid. The regenerated plants of both J. roemerianus and J. gerardi grew vigorously in potting soil in the greenhouse. J. roemerianus regenerants also grew well in a saltwater-irrigated field plot. Tissue culture-produced plants of J. roemerianus and J. gerardi can be used for planting in created or restored wetlands.