In vitro regeneration of the important North American oak species <Emphasis Type="Italic">Quercus alba</Emphasis>, <Emphasis Type="Italic">Quercus bicolor</Emphasis> and <Emphasis Type="Italic">Quercus rubra</Emphasis>

North American oak species, with their characteristic strong episodic seasonal shoot growth, are highly problematic for clonal micropropagation, resulting in the inability to achieve a stabilized shoot multiplication stage. The potential for initiating and proliferating shoot cultures derived from Quercus alba, Q. bicolor and Q. rubra explants was investigated, and a micropropagation method for these species was developed. Branch segments from 6 to 7-year-old trees were forced-flushed and the forced shoots were used as source of explants for culture initiation. A consistent shoot multiplication stage was achieved, in 13 of the 15 genotypes established in vitro, although marked differences occurred in explants from different genotypes/species. The control of efficient shoot multiplication involved the culture of decapitated shoots in a stressful horizontal position on cytokinin-containing medium with a sequence of transfers within a 6-week subculture cycle, which was beneficial to overcoming the episodic character of shoot growth. During each subculture cycle, the horizontally placed explants were cultured on media containing 0.2 mg l⁻¹ benzyladenine (BA) for 2 weeks with two successive transfers (2 weeks each) to fresh medium with 0.1 mg l⁻¹ BA, giving a 6-week subculture cycle. The general appearance and vigor of Q. alba and Q. bicolor shoot cultures were improved by the inclusion of both 0.1 mg l⁻¹ BA and 0.5 mg l⁻¹ zeatin in the medium used for the second transfer within the 6-week subculture cycle. Addition of AgNO₃ (3 mg l⁻¹) to the shoot proliferation medium of Q. rubra had a significant positive effect on shoot development pattern by reducing deleterious symptoms, including shoot tip necrosis and early senescence of leaves. The three species showed acceptable in vitro rooting rates by culturing microcuttings in medium containing 25 mg l⁻¹ indolebutyric acid for 48 h with subsequent transfer to auxin-free medium supplemented with 0.4% activated charcoal. Although an initial 5-day dark period generally improved the rooting response, it was detrimental to the quality of regenerated plantlets. However, activated charcoal stimulated not only the rooting frequencies, but it also enhanced plant quality, as evidenced by root, shoot and leaf growth.     

RAPD analysis of a variant of banana (Musa sp.) cv. grande naine and its propagation via shoot tip culture

Summary A morphological variant obtained from in vitro corm-derived plants of banana (Musa sp.) cv. Grande Naine (AAA) was evaluated up to harvest and the genetic basis of variation was confirmed by the random amplified polymorphic DNA (RAPD). The corms formed during the multiplication phase of shoot tip-derived cultures of the cv. Grande Naine grown on Murashige and Skoog (MS) medium enriched with 13.3 μM N ⁶-benzyladenine (BA) developed numerous morphological variants after transfer to MS medium with 6.66 μM BA. The variant designated as CUDBT-B1, with distinct morphological features, was further evaluated. The morphological features of CUDBT-B1 were variegated leaf, pseudostem, bracts, ovary of the male flower and fruits, reduced height, decreased lamina length and breadth, and early flowering. These features were also manifested in the second-cycle progeny of CUDBT-B1. RAPD assay showed a marker DNA band of 1650 bp, and differential band intensity between the CUDBT-B1 and normal clone. CUDBT-B1 was multiplied using shoot tip culture, and the shoots were rooted on half-strength MS medium supplemented with 2.69 μM α-naphthaleneacetic acid. All plantlets showed variegated leaves under field conditions.     

In vitro differentiation and plant regeneration of Albizia chinesis (Osb.) Merr

Summary Petiolar and distal cotyledonary segments (PCS and DCS) of Albizia chinensis were cultured on Murashige and Skoog's (MS; 1962) medium and induced to form adventitious shoot buds in the presence of either cytokinins 6-benzylamino purine (BAP), kinetin (KN) or thidiazuron (TDZ). Superiority of BAP in inducing shoot bud and differentiation was observed. PCS was more morphogenic to shoot bud differentiation than DCS. TDZ was highly effective in inducing shoot buds, but arrested shoot growth, while KN produced more callus during differentiation of shoots. Rapid and high rate of shoot multiplication per explant was achieved through subculture in MS medium containing BAP (1.0 mg l⁻¹) and indole-3-acetic acid (IAA) (0.5 mg l⁻¹). BAP at low concentration was required to enhance shoot multiplication and elongation. Successful rooting of regenerated shoots was carried out in a two-step culture procedure in MS media with indole-3-butyric acid (IBA) (2.0 mg l⁻¹) and subsequent subculture in IBA-free medium.     

Antioxidants enhance in vitro plant regeneration by inhibiting the accumulation of peroxidase in Virginia pine (<Emphasis Type="Italic">Pinus virginiana</Emphasis> Mill.)

Plant tissue necrosis and subsequent cell death are usually observed during in vitro regeneration in conifers, especially in plant regeneration via somatic organogenesis in pine species. Cell death is correlated with the elevated levels of peroxides. In this investigation, the effects of antioxidants on in vitro regeneration of Virginia pine (Pinus virginiana Mill.) were evaluated. Antioxidants, polyvinylpolypyrrolidone (PVPP) and 1,4-dithio-dl-threitol (DTT), were found to improve callus formation, shoot differentiation and growth, and shoot rooting by inhibiting tissue necrosis during the initiation of cultures and subculture of shoots. These treatments enabled the recovery and regeneration plants at high frequency through somatic organogenesis. Compared to the control, the frequencies of callus formation, shoot growth, and shoot rooting increased 15, 26, and 19%, respectively, by addition of 5 g/l PVPP and 2 g/l DTT. Higher peroxidase activity of tissue cultures during subculture from callus proliferation medium to shoot differentiation medium and to rooting medium was observed. The addition of antioxidants reduces and inhibits browning by reducing the accumulation of peroxidase.Abbreviations BA 6-Benzylaminopurine - 2,4-D 2,4-Dichlorophenoxyacetic acid - DTT 1,4-Dithio-dl-threitol - IBA Indole butyric acid - NAA -Naphthaleneacetic acid - PVPP Polyvinylpolypyrrolidone     

Acidogenic growth model of embryogenic cell suspension culture and qualitative mass production of somatic embryos from triploid bananas

A young male flower-derived embryogenic suspension cell population of AAA ‘Pei Chiao’, ‘Dwarf Cavendish’, and AAB ‘Raja’ was used for developing an acidogenic growth model . We hypothesized that a close relationship exists between the self-regulated pH medium and the corresponding changes in the growth phases. Studies have reported that a pH below 4.6 may prevent the embryogenic cells from undergoing polar growth. Controlling pH up to a level 4.6 within 2 days during the changes of pre-embryogenic cells (PECs) and proembryogenic masses into embryogenic determined cells (EDCs) uniformly resulted in unequal cell division. The hydrogen ion buffer 2-N-morpholino-ethanesulfonic acid at 10 g L⁻¹ was added to MA2 and MA3 media, showing the medium pH of MA3 up to 5.0, thus maintaining a relatively stable pH in AAA ‘Pei-Chiao’ and AAB ‘Raja’ cells that autoregulate acidification, significantly increasing the number of somatic embryos. When the proliferation and globularization phases were acidified to pH 3.5 ± 0.2, cells were released to free single cells of PECs and EDCs after 21 days. This study provides possible explanation that PECs deposit callose on their cell walls as a possible protector from strong acidic condition. Regulation at pH 5.0 ± 0.2 resulted the production efficiency achieved was 0.9 million somatic embryos per 1 mL of the settled cell volume.

     

Micropropagation of Calluna vulgaris cv. ‘H.E. Beale’

Axillary shoot producing cultures were obtained from microcuttings and shoot tips of Calluna vulgaris cv. H.E. Beale. For cultures derived from microcuttings the highest multiplication rate of 38 shoots (5 mm or longer) was obtained on a reduced salt medium with the addition of 0.5 mgl^-1 2-isopentenyladenine (2iP) during an 8 week subculture. For shoot tip derived cultures 0.2 mgl^-1 6-benzyladenine (BA) was the best cytokinin and led to a multiplication rate of 26 for a 6 week subculture. The addition of 1 g/l casein hydrolysate to a multiplication medium enhanced shoot proliferation in presence of 0.5 mgl^-1 BA.Despite various auxin treatments shoots formed no roots in vitro but rooted readily if transferred to a peat substrate ex vitro. A high rooting percentage (80%) was also obtained with shoots taken from the end of a multiplication phase and rooted directly. An additional subculture on low auxin containing media before transfer to peat substrate is recommended because the shoot condition can be improved in this way. A high number of rooted plantlets was produced, so the methods described will allow mass propagation.     

Mother plant age and seasonal influence on in vitro propagation of <Emphasis Type="Italic">Quercus euboica</Emphasis> Pap., an endemic,rare and endangered oak species of Greece

A micropropagation method for Quercus euboica Pap. was developed. Nodal explants from seedlings gave higher multiplication rates than explants from adult plants. Cultures initiated at the beginning of May produced the highest percentage of shoot forming explants and multiplication rate. Woody Plant Medium (WPM) salts, with 100 mg l⁻¹ myoinositol, 1 mg l⁻¹ thiamine, 0.5 mg l⁻¹ pyridoxine, 0.5 mg l⁻¹ nicotinic acid and 3% sucrose was used as basal medium and several cytokinins at various concentrations were evaluated for their effect on shoot multiplication. The highest shoot multiplication rate was obtained with 4.44 μΜ BA. IBA at 9.84 μΜ in the culture medium during the first week of culture, and if followed by culture in hormone-free medium, gave the best rooting results. Darkness at the beginning of the rooting period did not improve rooting. The use of plastic wrap as a cover material of the culture vessels enhanced rooting percentage and root number. Plantlets acclimatized ex vitro in soil from the natural environment of the species survived at a higher percentage (up to 93%) and had more vigorous growth than plantlets grown in a compost–perlite (2:1 v/v) medium (up to 36%).     

<Emphasis Type="Italic">In vitro</Emphasis> micropropagation and rooting of <Emphasis Type="Italic">Acacia mangium</Emphasis> microshoots from juvenile and mature origins

Acacia mangium microshoots from juvenile and mature genotypes were micropropagated through a regular subculture regime for more than 3 yr in vitro. Average multiplication rates of 5.5 for the juvenile source and of 3.9 for the mature clone were obtained during this period on the 6-benzylaminopurine-enriched multiplication medium. Although the juvenile material displayed higher potential for axillary shoot and root formation than the mature clone overall, the differences were not statistically significant with noticeable variations in the course of time from one subculture to another. On specific rooting media, the juvenile material rooted overall in greater proportions than the mature material, notwithstanding noteworthy interactions between the age of the plant material and the various experimental factors tested, i.e. sucrose concentration, macrosalt formulation and light regime. The stimulating effect of darkness on juvenile plant material rooting rates was more obvious than for the mature clone, which responded more inconsistently. Addition of 4 μM indole-3-acetic acid, indole-3-butyrie acid, or 1-naphthaleneacetic acid in the rooting medium significantly increased the proportion of rooted microshoots of both origins. The rooting criteria observed were also prone to vary depending on the experimental date. The data indicate that rooting of juvenile and mature Acacia mangium materials have average rates of 90% and 77%, respectively. These are high enough to consides possible applications of these procedures toward operational activities.     

Factors affectingin vitro shoot proliferation ofMyrtus communis L.: A comparison of adult and seedling material

Summary Two stocks of shoots growingin vitro, obtained from either seedlings or adult plants, were used to study the effects of material origin, the number of previous subcultures on the establishment medium, the explant type, and the macronutrients on shoot multiplication and elongation inMyrtus communis L., always in the presence of 4.4. μM benzyladenine (BA). Shoot proliferation was influenced mainly by stock origin, with higher responses from the adult material than from the seedling material, and by the number of subcultures, with the largest rates of multiplication and elongation in the first subculture. In the first subculture, the adult material was characterized by high rates of shoot multiplication and shoot elongation, and some shoots were hyperhydric. On the other hand, in the first subculture the seedling material was characterized by lower rates of shoot multiplication and elongation, and some shoots were affected by apical necrosis. In the third and the fifth subcultures, shoot multiplication and elongation declined in both materials, and hyperhydricity or apical necrosis were never found, although higher multiplication and elongation were consistently found for the adult material. The influence of the studied sources of variation is discussed in relation to shoot multiplication and elongation.     

In vitro root induction in axillary microshoots of Quercus robur L.

Whilst considerable efforts have been made to optimise shoot multiplication and rooting in oak, little attention has been paid to the impact of conditions used for multiplication on subsequent root formation. An optimised technique for rooting of oak microshoots has been developed to assess the effect of cytokinin treatments applied to shoot multiplication cultures on the subsequent rooting of microshoots. We found IBA to be more effective at inducing root formation in microshoots than NAA. Efficient rooting of oak microshoots (80%) was achieved after 35 days on medium supplemented with 1.0 mg litre^-1 IBA. Lower concentrations of IBA reduced the frequency of root formation and significantly increased the time taken for microshoots to form roots. High concentrations of IBA (3.0 mg litre^-1) produced similar rooting frequencies but with significantly increased numbers of roots formed by each microshoot. However, high concentrations of IBA stimulated the production of basal callus. Rooting of microshoots was unaffected by the concentration of BA used during shoot multiplication, although basal callusing was greater in microshoots taken from multiplication medium supplemented with the highest concentration of BA (1.0 mg litre^-1) and rooted on medium supplemented with 3.0 mg litre IBA. Reducing the period of exposure to auxin to 7 days by transferring microshoots to auxin-free medium increased the frequency of root formation (84%), led to more rapid root formation and a reduction in basal callus formation.     

CRISPR/Cas9 directed editing of lycopene epsilon-cyclase modulates metabolic flux for β-carotene biosynthesis in banana fruit

Banana is one of the most economically important fruit crops worldwide. Genetic improvement in banana is a challenging task due to its parthenocarpic nature and triploid genome. Genetic modification of crops via the CRISPR/Cas9 module has emerged as a promising tool to develop important traits. In the present work, a CRISPR/Cas9-based approach was used to develop the β-carotene-enriched Cavendish banana cultivar (cv.) Grand Naine (AAA genome). The fifth exon of the lycopene epsilon-cyclase (LCYε) gene was targeted. The targeting specificity of the designed guide-RNA was also tested by its ability to create indels in the LCYε gene at the A genome of cv. Rasthali (AAB genome). Sequence analysis revealed multiple types of indels in the genomic region of Grand Naine LCYε (GN-LCYε). Metabolic profiling of the fruit pulp of selected edited lines showed enhanced accumulation of β-carotene content up to 6-fold (~24 μg/g) compared with the unedited plants. These lines also showed either an absence or a drastic reduction in the levels of lutein and α-carotene, suggesting metabolic reprogramming, without any significant effect on the agro-morphological parameters. In addition, differential expression of carotenoid pathway genes was observed in the edited lines in comparison to unedited plants. Overall, this is the first report in banana to improve nutritional trait by using a precise genome editing approach.     

Aggressiveness and Damage Potential of Central American and Caribbean Populations of Radopholus spp. in Banana

Monoxenic cultures of burrowing nematode populations extracted from banana roots from Belize, Guatemala, Honduras, and Costa Rica were established on carrot discs. Cultures of Radopholus spp. were also obtained from Florida, Puerto Rico, Dominican Republic, and Ivory Coast. The aggressiveness (defined as reproductive fitness and root necrosis) of these populations was evaluated by inoculating banana plants (Musa AAA, cv. Grande Naine) with 200 nematodes/plant. Banana plants produced by tissue culture were grown in 0.4-liter styrofoam cups, containing a 1:1 mix of a coarse and a fine sand, at ca. 27 °C and 80% RH. Banana plants were acclimated and allowed to grow for 4 weeks prior to inoculation. Plant height, fresh shoot and root weights, root necrosis, and nematode population densities were determined 8 weeks after inoculation. Burrowing-nematode populations varied in aggressiveness, and their reproductive fitness was generally related to damage reported in the field. Plant height and fresh shoot and root weight did not reflect damage caused by nematodes under our experimental conditions. Necrosis of primary roots was closely related to the reproductive fitness of the nematode populations. Variation in aggressiveness among nematode populations followed a similar trend in the two susceptible hosts tested, Grande Naine and Pisang mas. All nematode populations had a low reproductive factor (Rf ≤2.5) in the resistant host except for the Ivory Coast population which had a moderate reproductive factor (Rf ≤ 5) on Pisang Jari Buaya. This is the first report of a burrowing nematode population parasitizing this important source of resistance to R. similis.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of <Emphasis Type="Italic">Gentiana dinarica</Emphasis> Beck.

Gentiana dinarica Beck, rare and endangered species of Balkan Dinaric alps, was in vitro propagated (micropropagated) from axillary buds of plants collected at Mt. Tara, Serbia. G. dinarica preferred MS to WPM medium, with optimal shoot multiplication on MS medium with 3% sucrose, 1.0 mg l⁻¹ BA and 0.1 mg l⁻¹ NAA. Rooting was not clearly separated from shoot multiplication since BA did not completely inhibit root initiation. Spontaneous rooting on plant growth regulator-free medium occurred in some 30% of shoot explants. Rooting was stimulated mostly by decreased mineral salt nutrition and a medium with 0.5 MS salts, 2% sucrose and 0.5–1.0 mg l⁻¹ IBA was considered to be optimal for rooting. Rooted plantlets were successfully acclimated and further cultured in peat-based substrate.     

In vitro plantlet regeneration of “dwarf” Indian olive (Elaeocarpus robustus Roxb.): a fruit plant of Bangladesh

A plantlet regeneration protocol was developed on pot-grown mature plants of Elaeocarpus robustus Roxb. cv. Dwarf from nodal and leaf explants. The best yield of adventitious shoots was achieved from the leaf-derived calli in a modified MS (MMS₁, half strength of major salts, full strength of minor salts, and vitamins) medium containing 4.0 μM BA + 4.0 μM Kn + 0.5 μM NAA + 15% coconut water (CW). The shoot multiplication rate was amplified about twofold per culture after the addition of 15% CW to the medium. The rate of shoot multiplication reached maximum at the 5th subculture, and it maintained this rate throughout the 3 subsequent subcultures. The best rooting in vitro was investigated by subculturing the microcuttings in an MMS₂ (half strength of both major salts and minor salts and full strength of vitamins) medium containing 1.0 μM IBA in the dark for one initial week at 30°C, followed by subculturing them in a plant-growth regulator (PGR)-free medium in the light. The plantlets raised in vitro were successfully established under ex vitro conditions.     

Allelopathic effects of Cyperus rotundus extract in vitro and ex vitro on banana

Allelopathic effects of Cyperus rotundus on banana ‘Grande Naine’ were studied in vitro as well as ex vitro. To study the allelopathic effects in vitro, the Cyperus extract was added to the multiplication medium during preparation before adjusting the pH. Of the four concentrations, 0.2 and 0.6% decreased the shoot multiplication and shoot length but 1 and 2% extract completely inhibited the shoot multiplication but induced rooting in 50 and 28% shoots, respectively. For the ex vitro studies, the Cyperus extract was added to the hydroponic medium during the hardening of in vitro raised banana plantlets. The extract of 1 and 2% concentrations decreased the shoot and root length, number of leaves and new roots, fresh weight, total chlorophyll and protein. The fluorescence intensity ratio was successively increased resulting in decreased photosynthesis.     

In vitro Storage of Strawberry and Raspberry in Calcium-Alginate Beads at 4°C

Three-millimeter-long shoot tips of strawberry 'Senga Sengana' and raspberry 'Norna' encapsulated in calcium alginate were stored in vitro at 4 °C in the dark. The cultures which were donors for the shoot tips were grown before encapsulation on shoot multiplication media (Boxus medium with 2.2 µM BAP and 2.46 µM IBA for strawberry, and MS medium with NH₄NO₃ and KNO₃ reduced by 50%, and with 3.55 µM BAP and 0.49 µM IBA for raspberry) as well as on these media supplemented with 10 g l^–1 mannitol or paclobutrazol (1.7 µM for strawberry and 3.4 µM for raspberry). Sodium alginate was dissolved in water, water with sugar or in a culture medium without growth regulators. Regrowth ability of the stored explants and in vitro multiplication in three successive subcultures were evaluated. The encapsulated shoot tips could be stored for 9 months in beads containing sugar or a culture medium. The pre-conditioning of the donor cultures on a mannitol containing medium was beneficial for regrowth ability. The multiplication rate of strawberry and raspberry shoots in the first subculture after storage was lower than that of non-stored cultures. Particularly low multiplication was obtained for strawberry which had been stored for 9 months and for raspberry stored for 3 and 6 months, in combinations where the beads were prepared by dissolving sodium alginate in water. Multiplication of strawberry in the second subculture was generally higher than in non-stored cultures, but multiplication of raspberry was lower also in the second subculture, with the exception of the combination stored for 9 months and pre-cultured on mannitol. In the third subculture, shoot multiplication in both species was similar to that in non-stored cultures.     

Thidiazuron (TDZ) induced organogenesis and clonal fidelity studies in <Emphasis Type="Italic">Haloxylon persicum</Emphasis> (Bunge ex Boiss &amp; Buhse): an endangered desert tree species

Haloxylon persicum (Bunge ex Boiss & Buhse), is one of the hardy woody desert shrubs, which is now endangered and/or nearing extinction. Urban landscape development and overgrazing are the major threats for the erosion of this important plant species. For conserving the species, it is critical to develop an efficient in vitro regeneration protocol for rapid multiplication of large number of regenerants. Leaf explants, cultured on Murashige and Skoog (MS) medium supplemented with different concentrations of thidiazuron (TDZ) (0.5, 1, 2 µM), showed significant difference in bud sprouting and adventitious shoot induction. The highest shoot bud formation was recorded on MS medium supplemented with 0.5 µM TDZ. Shoot tip necrosis (STN), observed after first subculture of shoot buds in same medium, increased in severity with subculture time. Application of calcium (4 mM) and boron (0.1 mM) in combination with kinetin (10 µM) in the subculture medium significantly reduced the intensity of STN. On an average eight shoots/explant were produced by alleviating this problem. ISSR marker analysis revealed monomorphic banding pattern between progenies and parents, indicating the true to type nature of the clones and its parents.     

Anatomical and Biochemical Changes Associated with In Vitro Rhizogenesis in Dendrocalamus giganteus

Successful micropropagation protocol of a difficult-to-root bamboo species, Dendrocalamus giganteus (10–15 years old) along with the analysis of anatomical and biochemical changes during in vitro rhizogenesis was accomplished. Proliferated axillary shoots from nodal segments of 10–15 years old field culms exhibited shoot necrosis during multiple shoot formation phase and was controlled by subculturing in modified MS liquid medium having 825 mg ^l?1 NH₄NO₃, 3800 mg ^l?1 KNO₃, 740 mg ^l?1 MgSO₄ and 9% coconut water, 26.64 μM 6-benzylaminopurine (BA) and 0.46 μM kinetin. These multiple shoots proliferated from field grown culms, failed to root and hence callus was induced on MS solid medium containing 4.44 μM BA, 4.52 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 5.37 μM naphthalene acetic acid (NAA). Organogenesis from the callus was achieved upon transfer to MS medium with 11.10 μM BA and 2.32 pM kinetin. The callus-derived shoots multiplied on modified MS medium were rooted the best (91%) by culturing 3 days on MS medium having glucose (0.5%), sucrose (2.5%) and 98.41 μM indolebutyric acid (IBA) and subsequently to IBA-free MS medium containing 3% sucrose. Studies on peroxidase and IAA oxidase activity and endogenous free- and bound-IAA content showed that IAA oxidase and peroxidase oxidize endogenous IAA resulting in root initials formation. Anatomical studies confirmed the root primordia formation from 3^rd day of IBA treatment and primordia were visible over the surface on 8^th to 10^th day. However, the shoot necrosis symptoms which started on 6^th day of treatment intensified by 10^th day leading to the death of the whole shoot system by 12^th–15^th day. Nevertheless, on the root formation medium with 9.84 μM IBA, new shoot buds were emerged and showed shoot growth in 60% of the rooted cultures, which were successfully acclimatized in shade-house with 100% survival. The present study establishes rooting of callus-derived shoots as the best way for the successful propagation of the difficult-to-root bamboo, D. giganteus when compared to axillary bud proliferated shoots.     

葡萄柚种子无菌苗组培快繁体系的建立

以葡萄柚种子在无菌条件下萌发的幼苗作为外植体供体,子叶切断作为丛生芽初代诱导培养的接种材料,以MT作为基本培养基,通过调节不同植物生长调节剂及其浓度组合、不同质量浓度的蔗糖,选择最佳初代培养基、继代培养基和生根培养基,对葡萄柚组培快繁技术体系进行研究。结果表明:(1)葡萄柚种子经预处理后,先用75%酒精表面灭菌15 s,再用0.1%Hg Cl2浸泡20 min的消毒效果最好,污染率为18.33%,萌发率为89.91%;(2)初代培养的最适培养基为MT+1.0 mg·L-16-BA(6-苄氨基腺嘌呤)+0.2 mg·L-1IBA(吲哚丁酸)+0.2 mg·L-1GA(赤霉素)+蔗糖40 g·L-1,腋芽诱导率较高,为93.33%;(3)浓度为0.2 mg·L-1的GA能提高初代培养的腋芽诱导率,但还达不到显著水平;(4)继代培养的最适培养基为MT+0.5 mg·L-16-BA+0.2 mg·L-1IBA+0.2 mg·L-1GA+蔗糖40 g·L-1,丛生芽增殖系数达到4.25,芽深绿色,茎粗壮,节间较长,生长旺盛;(5)浓度为0.2 mg·L-1的GA能显著提高继代培养的增殖系数;(6)最适合继代培养的蔗糖浓度为40 g·L-1,丛生芽长势极好;(7)最佳生根培养基为1/2 MT+NAA 0.2 mg/L+蔗糖40 g·L-1+AC 0.1%,生根率达68.89%,根粗壮;(8)生根苗移栽30 d后成活率在75%以上。该研究建立了葡萄柚的组织培养快繁技术体系,为葡萄柚的规模化生产提供了可行的技术依据。     

Comparative studies of cytokinins on in vitro propagation of Bacopa monniera

A mass in vitro propagation system for Bacopa monniera (L.) Wettst. (Scrophulariaceae), a medicinally important plant, has been developed. A range of cytokinins have been investigated for multiple shoot induction with node, internode and leaf explants. Of the four cytokinins (6-benzyladenine, thidiazuron, kinetin and 2-isopentenyladenine) tested thidiazuron (6.8 μM) and 6-benzyladenine (8.9 μM) proved superior to other treatments. Optimum adventitious shoot buds induction occurred at 6.8 μM thidiazuron where an average of 93 shoot buds were produced in leaf explants after 7 weeks of incubation. However, subculture of leaf explants on medium containing 2.2 μM benzyladenine yielded a higher number (129.1) of adventitious shoot buds by the end of third subculture. The percentage shoot multiplication (100%) as well as the number of shoots per explant remained the high during the first 3 subculture cycles, facilitating their simultaneous harvest for rooting. In vitro derived shoots were elongated on growth regulator-free MS medium and exhibited better rooting response on medium containing 4.9 μM IBA. After a hardening phase of 3 weeks, there was an almost 100% transplantation success in the field. This revised version was published online in June 2006 with corrections to the Cover Date.