Plant regeneration from leaf explants of Aloe barbadensis Mill. and genetic fidelity assessment through DNA markers

An efficient plant regeneration protocol was developed from leaf explants of Aloe barbadensis Mill on Murashige and Skoog’s (MS) medium supplemented with 2.0 mg/l 6-benzyladenine (BA) or Kinetin (Kn), 0.25–0.5 mg/l NAA (1-napthalene acetic acid) and 3 % (w/v) sucrose within 4 weeks of culture. The maximum number of shoot buds were obtained on MS medium supplemented with 2.0 mg/l BA, 0.5 mg/l NAA, 40 mg/l Ads (adenine sulphate) within 4–6 weeks of subculture. Inclusion of 0.25–0.50 mg/l gibberellic acid into the medium, the shoot buds became elongated. Repeated subculture on regeneration medium induces higher rate of shoot regeneration. The root induction from excised microshoots was achieved on half-strength MS medium supplemented with 0.25–1.0 mg/l NAA or indole-3-butyric acid (IBA) and 2 % (w/v) sucrose. Maximum percentage of rooting was achieved on medium having 0.5 mg/l NAA with 3 % (w/v) sucrose. About 80 % of in vitro raised plantlets were hardened in the greenhouse and successfully established in the soil. Both Random Amplified Polymorphic DNA (RAPD) and Inter Simple Sequence Repeat (ISSR) markers were used to detect the variability among the regenerated plants developed in vitro. The results showed that there was no polymorphism among the regenerated plantlets. This study will help for propagation of quality planting material of Aloe barbadensis for commercialization.     

Somatic embryogenesis and in vitro flowering of 3 species of bamboo

Plant regeneration via somatic embryogenesis was achieved in callus cultures derived from nodal explants of in vitro grown seedlings and excised mature zygotic embryos of three bamboo species on Murashige and Skoog's (MS) basal medium supplemented with 0.5 mg/l kinetin (Kn), 2.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), 10 mg/l adenine sulphate (Ads) and 3% (w/v) sucrose incubated in the light or in the dark. Somatic embryos germinated (95–98%) into normal plants and were transferred to soil with 95% success. In vitro flowering was induced on shoots developed from nodal explants taken from somatic embryo regenerated plants of Bambusa vulgaris, Dendrocalamus giganteus and Dendrocalamus strictus on half-strength MS basal medium supplemented with 0.25 mg/l indole-3-butyric acid (IBA), 0.5 mg/l Ads, 0.5 mg/l gibberellic acid (GA₃) and 3% sucrose.Abbreviations BAP 6-benzylaminopurine - Kn kinetin - Ads adenine sulphate - IBA indole-3-butyric acid - NAA 1-naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog (1962) basal medium - GA₃ gibberellic acid     

<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.     

High frequency shoot regeneration from nodal and shoot tip explants in Holarrhena antidysenterica L

Shoot tip and nodal segment explants of Holarrhena antidysenterica when cultured on MS medium containing BAP (1.0-3.0 mg/l) with NAA (0.2-1.0 mg/l) and BAP (1.0-3.0 mg/l) with Kn. (0.2-1.0 mg/l) produced multiple shoots. Maximum multiple shoots was found in MS medium supplemented with BAP (2.0 mg/l) and NAA (0.5 mg/l). Subculture on the same medium resulted in rapid shoot multiplication at an average rate of 16 new shoots per subculture. Addition of urea (100 mg/l) in the medium increased the number of shoots up to 22 per culture. For best rooting, the shoots were excised from the culture flask and implanted individually on half strength MS medium with 0.5 mg/l each of IBA, IAA and NAA. After 20 days of transfer on root induction medium 95% rooting was achieved. Regenerated plantlets were successfully acclimatized and established in soil. About 90% of plantlets survived under open field conditions.     

In vitro somatic embryogenesis from callus culture of the timber yielding treeHardwickia binata Roxb.

Somatic embryogenesis was achieved in callus cultures derived from immature cotyledonary explants ofHardwickia binata Roxb., a multipurpose leguminous tree, on semisolid modified Murashige and Skoog's (mMS) medium containing 2900 mg/l potassium nitrate (KNO₃) supplemented with 4.64 µM kinetin (Kn) and 5.37µM a-naphthaleneacetic acid (NAA). Somatic embryos proliferated rapidly after transfer to MS basal medium supplemented with 2052.6 µM L-glutamine and 0.084 µM gibberellic acid (GA₃). Maturation of somatic embryos was achieved on half-strength MS basal medium supplemented with 1.23 µM IBA and 2% (w/v) sucrose. Histological studies confirmed different developmental stages of somatic embryogenesis inHardwickia binata. Abbreviations BA N⁶-benzyladenine - Kn kinetin - NAA a-naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - IBA indole-3-butyric acid - GA₃ gibberellic acid - MS Murashige and Skoog (1962) medium - mMS modified Murashige and Skoog (1962) medium     

The Effect of Plant Growth Regulators and Sucrose on the Micropropagation and Microtuberization of <Emphasis Type="Italic">Dioscorea nipponica</Emphasis> Makino

The effects of sucrose, plant growth regulators, MS (Murashige and Skoog), and ½MS salt media formulations were investigated for the development of shoot cultures, microtuber induction, and plantlet regeneration in Dioscorea nipponica. The cytokinin N-benzyladenine (BA) in the range of 0.5–2.0 mg/l showed strong enhancing effects on microtuber induction only when used in conjunction with the auxin alpha-naphthalene acetic acid (NAA), with the effect that NAA increased from 0.5 to 2.0 mg/l. Murashige and Skoog salt media supplemented with sucrose at 3% (w/v) gave the highest frequencies of shoot induction (86%) when BA was present at 2.0 mg/l and NAA at 1.0 mg/l. Sucrose at 7% (w/v) was the single most significant medium constituent for microtuber growth. The heaviest microtubers were formed on media containing 1.0 mg/l BA and 2.0 mg/l (0.073 g), especially with 7% sucrose (3.46 g). With media containing ½MS, 2% sucrose, and 0.1% (w/v) activated charcoal, the percentage of rooting was maximal when supplemented with 1.0 mg/l BA and 0.5 mg/l NAA for the in vitro produced shoots (95%) and BA and NAA both at 0.5 mg/l for the microtubers (100%). When removed from culture flasks and transferred into sterilized soil in a greenhouse, most of the hardened plantlets survived (over 91% after 1 week), and they were suitable for field planting after 1 month.     

In Vitro Multiplication of Beta Vulgaris L. throughout Excised Shoot Tips

Friable calli were induced from mature excised shoots of Bambusa vulgaris on Murashige and Skoog's (MS) medium supplemented with 2.2 μM6-benzylamino-purine (BAP), 9.04 μM 2,4-dichlorophenoxyacetic acid and 14.76 μM indole-3-butyric acid (IBA) with 3 % (m/v) saccharose. Adventitious shoots with root hairs were achieved from calli on MS medium supplemented with 13.33 μM BAP and 1.23 - 2.46 μM IBA within 4 weeks of subculture. The frequency of shoot bud regeneration was better in the light incubated cultures than in the dark incubated cultures. Isolated shoots were rooted on liquid half-strength MS basal medium supplemented with 0.49 μM IBA and 2 % (m/v) saccharose. Histological observations confirmed the regeneration of shoot buds from calli. The rooted plantlets were successfully transferred to greenhouse. This revised version was published online in July 2006 with corrections to the Cover Date.     

In vitro micropropagation of Lawsonia inermis (Lythraceae)

A successful protocol was developed for mass propagation of Lawsonia inermis Linn., an important medicinal plant. Multiple shoots were induced in apical and axillary meristems derived from mature explants of L. inermis on Murashige and Skoog (1962) medium supplemented with 0.25 mg/l 6-benzylaminopurine (BA), 0.25 mg/l Kinetin (Kn), 0.5 mg/l ascorbic acid and 3% (w/v) sucrose. The rate of multiplication was higher when the cultures were incubated under continuous light rather than the 14 hr photoperiod. Rooting was readily achieved upon transferring the microshoots onto MS basal semi-solid medium supplemented with 0.25 mg/l indole-3-butyric acid (IBA) after ten days of culture. Micropropagated plantlets were acclimatized and successfully grown in soil.     

High frequency of shoot multiplication and bulblet formation of garlic in liquid cultures

In vitro shoot proliferation and bulblet production of garlic (Allium sativum L.) was studied in liquid cultures. Shoots grown in vitro were used as explants and were cultured in MS medium supplemented with 2% (w/v) sucrose and 0.5 mg l^–1 2-iP. Three culture methods (semi-solid, liquid-immersion and raft) were compared for shoot proliferation. Explants in liquid (immersion) culture exhibited an increased multiplication rate and fresh weight of shoots after 3 weeks of culture as compared with the other treatments. Bulblet formation and growth were studied in liquid medium with different concentrations of sucrose (2–13%). MS medium containing 11% (w/v) sucrose was optimal for bulblet development and bulblets developed in this medium within 9 weeks in culture. The highest multiplication rate was (135 bulblets/explant) found when explants were cultured in bulbing medium (MS medium containing 0.1 mg l^–1 NAA+11% (w/v) sucrose) supplemented with 10 M JA. Growth retardants CCC, B-9, ABA also promoted induction and growth of bulblets. Darkness promoted the bulblet induction and growth compared to light conditions (16-h photoperiod of 50 mol m^–2 s^–1). The dormancy of bulblets was broken by cold treatment at 4 °C for 8 weeks.     

Rapid plant regeneration and analysis of genetic fidelity of in vitro derived plants of Chlorophytum arundinaceum Baker—an endangered medicinal herb

An efficient in vitro multiplication system via multiple shoot bud induction and regeneration has been developed in Chlorophytum arundinaceum using shoot crown explants. Optimum regeneration frequency (87%) and desirable organogenetic response in the form of de novo organized multiple shoot buds without an intervening callus phase was obtained on Murashige and Skoog's (MS) minimal organics medium containing 3% sucrose (w/v) supplemented with 4×10⁻⁶ M Kn and 2×10⁻⁶ MIBA. Axenic secondary explants with multiple shoot buds on subculturing elicited best response with 1×10⁻⁵ M Kinetin (Kn) and 5×10⁻⁶ M indole-3-butyric acid (IBA) giving rise to an average of 18.74 shoots per culture with mean shoot length of 7.6 cm ± 1.73. Varying molar ratios of either Kn/IBA or Kn/NAA revealed statistically significant differences in the regeneration frequencies among the phytohormone treatments. It was observed that the shoot bud differentiation and regeneration was influenced by the molar ratios of cytokinins/auxin rather than their relative concentrations. Healthy regenerated shoots were rooted in half strength MS basal medium containing 3% sucrose (w/v) supplemented with 5×10⁻⁶ M IBA. Following simple hardening procedures, rooted plantlets, were transferred to soil-sand (1:1; v/v) with more than 90% success. Genetic fidelity was assessed using random amplified polymorphic DNA (RAPD), karyotype analysis and meiotic behaviour of in vitro and in vivo plants. Five arbitrary decamers displayed same banding profile within all the micropropagated plants and in vivo explant donor. The cytological and molecular analysis complemented and compared well and showed no genomic alterations in the plants regenerated through shoot bud differentiation. High multiplication frequency, molecular, cytological and phenotypic stability ensures the efficacy of the protocol developed for the production and conservation of this important endangered medicinal herb.     

沙棘组织培养技术的研究

本试验选用中国沙棘的种子和休眠枝条作为材料,研究沙棘不同外植体的离体培养技术,试验结果表明,沙棘的无菌苗子与休眠枝条上的休眠芽都可以作为离体培养的良好外植体,沙棘的最适分化培养基是：1／4MS＋6－BA0．30mg/L NAA0．002mg/L,增殖,壮芽培养基是：1／4 MS＋6－BA 0．1mg/L NAA 0.004mg/L;最适生根培养基是：1／4MS＋NAA0.05mg/L iBA 0.2mg/L;愈伤组织诱导培养基是：1／4MS＋2,4－D 0．3mg/L。     

In vitro propagation of shoot buds of Carica papaya L. (caricaceae) var. Honey Dew

Shoot buds from the saplings and the fruit bearing plants of Carica papaya L.. var. Honey Dew (papaya) initially treated with Gentamycin were cultured in modified MS media, each with a different hormonal combination, for the establishment of cultures and multiplication and rooting of plants. About 43% of explants from fruit bearing plants and 69% of those from saplings remained free of contamination and retained regeneration capacity when treated in 500 mg/l Gentamycin. For the establishment of the explants a medium containing 1 mg/l GA₃ and 2 mg/l kinetin was necessary. When established buds were transferred to medium containing 1 mg/l NAA and 3 mg/l kinetin, calli were initiated at cut ends of shoot buds; multiplication started on transfer to NAA (0.1 mg/l) and BAP (0.5 mg/l) medium. Cultures have been maintained for the last twenty months without any loss in multiplication rate. Rooting was induced in medium with reduced salt concentration containing 2 mg/l IBA. Shoot elongation was induced after prolonged culture in the same rooting medium.Abbreviations MS Murashige and Skoog, 1962 - SH Schenk and Hildebrandt, 1972 - GA₃ Gibberellic acid - Kn Kinetin - NAA Napthaleneacetic acid - BAP 6 -Benzylaminopurine - IBA Indole-3-butyric acid - IAA Indole-3-acetic acid     

不同生长调节剂对狗肝菜愈伤组织诱导和离体快繁的影响

目的 :研究不同生长调节剂对狗肝菜愈伤组织诱导和离体快繁的影响。方法 :狗肝菜不同外植体在附加不同生长调节剂的培养基上诱导愈伤组织 ,比较愈伤组织的诱导率 ;用 3因子 5水平的正交实验 ,比较不同生长调节剂对丛生芽诱导的影响 ;在附加不同生长调节剂的培养基上比较芽增殖倍数 ;附加不同浓度NAA的培养基上比较生根效果。结果 :愈伤组织诱导率相对以叶片最高 ,茎段次之 ,最后为叶柄 ;愈伤组织诱导的最佳培养基为MS 6-BA0 .5 NAA1 .5 ;不同激素对茎段芽诱导的影响次序为 6-BA>KT >NAA ,芽诱导的最佳培养基为MS 6-BA2mg/L KT1mg/L NAA0 .5mg/L ;芽继代增殖的最佳激素组合是MS 6-BA2mg/L NAA2mg/L ,增殖倍数达 3.0 0 ,影响芽继代增殖的因素次序为 6-BA >NAANAA0 .5mg/L的生根效果较好。结论 :附加一定的生长调节剂能提高狗肝菜愈伤组织的诱导率和离体快繁的效率。     

倒挂金钟的离体快繁及其培养基简化试验

试验以倒挂金钟茎段为外植体建立了其离体再生体系,并对培养过程中的培养基简化进行了研究.结果表明:茎段增殖的最适培养基配方为Ms+6-BA(1.0 mg/L)+KT(0.5 mg/L)+NAA(0.2 mg/L);最适生根培养基为1/2 MS+NAA(0.5 mg/L)+AC(0.3%).在其大规模生产中,可用白砂糖代替蔗糖,自来水代替蒸馏水.移栽试验表明,4月份的移栽成活率比3月份明显提高,用蛭石:田园土(1:1)作基质,移栽成活率可达92.8%.     

In vitro propagation and rhizome formation in Curcuma longa Linn

Turmeric (Curcuma longa Linn.) which is cultivated by underground rhizomes is a slow propagating species. Multiplication and callus induction starting from the rhizome buds and shoot tips of C. longa in MS medium was carried out. A combination of naphthalene acetic acid (NAA; 1.0 mg/l) with kinetin (Kn; 1.0 mg/l) or NAA (1.0 mg/l) with 6-benzylaminopurine (BAP; 2.0 mg/l) was optimum for rapid clonal propagation of turmeric. A concentration of 2.5-3.0 mg/l of 2,4-dichlorophenoxy-acetic acid (2,4-D) was found to be optimum for callus induction. Regeneration of plantlets from a callus was successfully conducted in MS medium supplemented with standard growth hormones for multiplication at 25 +/- 2 degrees C under a 16 h photoperiod. These plantlets were successfully transferred to the field. Plantlets (4-month-old) were incubated in a medium containing different concentrations of sucrose supplemented with NAA (0.1 mg/l) and Kn (1.0 mg/l) at 27 +/- 2 degrees C under an 8 h photoperiod for induction of rhizomes. In vitro rhizome formation was observed in media containing 6 and 8% sucrose.     

荷兰鸢尾(Iris xiphium L. var. hybridum)的组织培养

取荷兰鸢尾（ＩｒｉｓｘｉｐｈｉｕｍＬ．ｖａｒ．ｈｙｂｒｉｄｕｍ）鳞茎片不同部位外植体块,接种于附加不同激素配比的基本培养基上,其中取自鳞茎片基部外植体块２ｍｍ×２ｍｍ×２ｍｍ,培养基为ＭＳ＋ＢＡ１．０ｍｇ／Ｌ＋ＮＡＡ０．２ｍｇ／Ｌ的不定芽诱导率为最高（７０％）;最理想的增殖培养基为ＭＳ＋ＢＡ２．０ｍｇ／Ｌ＋ＮＡＡ０．２ｍｇ／Ｌ。不定芽直径４～５ｍｍ,培养基为ＭＳ＋ＢＡ０．２ｍｇ／Ｌ＋ＮＡＡ０．５ｍｇ／Ｌ有利于不定根的发生,诱导生根率达８３３％。试管苗不经练苗可直接出瓶,移栽于泥炭∶田土∶河沙＝１∶１∶１（Ｖ／Ｖ）的基质中。     

Micropropagation ofFagus sylvatica L.

Summary An in vitro shoot multiplication system was established from juvenileFagus sylvatica L. tissues, and plantlets were regenerated. Embryonic axes were excised from beech seeds and germinated in vitro on media supplemented with 6-benzyladenine (BA) to obtain plantlets with axillary shoots. Shoot multiplication was maintained by sequential subculture of axillary shoot tips and basal segments on Woody Plant Medium supplemented with 0.5 mg/liter BA+2 mg/liter zeatin+0.2 mg/liter naphthaleneacetic acid (NAA). The effeciency of shoot multiplication clearly depended on the kind of explant used. Transfer to fresh medium every 2 wk during the 6-wk multiplication cycle improved multiplication rates. In the rooting stage, an initial 7-day dark period significantly improved rooting capacity and accelerated the emergence of roots on auxin-treated shoots. Adventitious buds were induced on the intact hypocotyls of the whole plantlets derived from the initial embryonic axis explants, especially on those cultured on medium with 1 mg/liter BA. Cotyledon and hypocotyl segments isolated from seedlings grown in vitro from embryos also exhibited capacity for adventitious bud formation, especially when cultured on media supplemented with 0.5 mg/liter BA + 0.1 mg/liter NAA.     

In vitro clonal propagation of Nyctanthes arbor-tristis

Rapid shoot multiplication of Nyctanthes arbor-tristis L. was achieved from axillary meristems on Murashige and Skoog (MS) basal medium supplemented with 1.0–1.5 mg dm⁻³ 6-benzylaminopurine (BA), 50 mg dm⁻³ adenine sulfate (Ads) and 3 % (m/v) sucrose. Inclusion of indole-3-acetic acid (IAA) in the culture medium along with BA + Ads promoted a higher rate of shoot multiplication. Maximum mean number of microshoots per explant (6.65) was achieved on the MS medium supplemented with 1.5 mg dm⁻³ BA, 50 mg dm⁻³ Ads and 0.1 mg dm⁻³ IAA after 4 weeks of culture. The elongated shoots rooted within 13 to 14 d on half-strength MS medium supplemented with either indole-3-butyric acid (IBA), IAA or 1-naphthaleneacetic acid (NAA) with 2 % sucrose. Maximum percentage of rooting was obtained on medium having 0.25 mg dm⁻³ IBA and 0.1 mg dm⁻³ IAA. About 70 % of the rooted plantlets survived in the greenhouse. The in vitro raised plants were grown normally in the field.     

Augmenting in vitro shoot multiplication by vipul (triacontanol) and adventitious rhizogenesis by rice bran extract in Dendrocalamus strictus

Like other bamboo species, Dendrocalamus strictus flowers gregariously after a prolonged intermast period of 48 years and constitutes an ideal material for in vitro clonal propagation. In this study, MS liquid medium containing 0.5, 1.0 and 2.0 mL/L vipul (Godrej Agrovet, Ltd., Sachin, India), a commercial formulation of triacontanol, with or without BA (3.0 mg/L) was tested for in vitro shoot multiplication and 1.0, 2.5 and 5.0 mL/L of 20% (w/v) alcoholic/aqueous rice bran extract (alone or in combination) with NAA (3 mg/L) used for in vitro adventitious rhizogenesis in single node culture derived shoots of Dendrocalamus strictus.. After a multiplication cycle for 4-5 week, vipul (0.5 mL/L) with BA (3.0 mg/L) in the culture medium induced 4.59 fold shoot multiplication rate whereas application of BA and vipul alone had corresponding values of 3.29 and 0.53 fold respectively. Maximum vipul concentration (2 mL/L) with BA (3 mg/L) exhibited shoot multiplication higher than (or equal to) that of BA alone. Maximum in vitro rooting percentage (55.66%) was obtained on half MS medium enriched with alcoholic rice bran extract (2.5 mL/L) and NAA (3 mg/L). This is the first investigation reporting amelioration of in vitro shoot multiplication rate by triacontanol and rooting percentage by rice bran extract in explants from mature bamboo culms. The protocol is economical and rapid for in vitro clonal propagation of Dendrocalamus strictus.     

Effect of age of seedling and phytohormones on micropropagation of indica rice (Oryza sativa L.) from Meristem Culture.

An efficient protocol forin vitro micropropagation of seven indica rice varieties was developed from meristem culture. Meristem (leaf base) was isolated from different age of seedlings and cultured on MS medium without hormones and supplemented with different concentrations of NAA and BAP. Regeneration of plantlets from meristem was observed within five days of culture. The meristem isolated from 4-day old seedlings gave highest regeneration on hormone free MS medium. Histological study of meristem (leaf base) from 4-day old seedlings confirmed the presence of meristematic cells. Regenerated plants were multiplied on MS medium supplemented with 0.05 mg/L NAA and 5 mg/L BAP. An average of five plants were obtained from single regenerated meristem. The plants regenerated from meristem showed morphological uniformity.