Micropropagation and Callus Culture of Saussurea laniceps, an Alpine Medicinal Plant

Cottonhead windhairdaisy (Saussurea laniceps Hand.-Mazz.) is one of the most famous and important medicinal herbs in China. Illegal collection from wild populations is increasingly threatening the present environment of S. laniceps. Estab- lishment of an efficient method for micropropagation is the best way to change its endangered situation. When mature seeds of S. laniceps were cultured on hormone-free MS medium, plantlets were formed from germinated seeds in 7–10 d. Then 0.5 cm × 0.5 cm leaf explants were transplanted to MS medium supplemented with 1-naphthalene-acetic acid (NAA)/2,4-D and benzyladenine (BA)/KT and callus was achieved 10 d after transfer. Shoot bud regeneration occurred from callus cultured on MS medium supple- mented with different growth regulators 20 d after culturing. The regeneration percentages varied with the different components of plant growth regulators. The percent regeneration from callus pretreated at low temperature of 5°C increased significantly compared with those incubated at 23/20°C directly. Optimal regeneration was observed with explants on media supplemented with 1.5 mg·L–1 BA plus 0.2 mg·L–1 NAA. In the presence of 0.2 mg·L–1 NAA in half-strength MS, 78% of the shoots formed roots. Plantlets from explants showed 63% survival after acclimatization.

<i>In Vitro</i>Micropropagation of a Valuable Medicinal Plant, <i>Plectranthus amboinicus</i>

The effect of the plant growth regulators benzyl amino purine (BAP), 1-naphthaleneacetic acid (NAA) and kinetin (KIN) on in vitro shoot induction and proliferation of Plectranthus amboinicus was examined. Explants obtained from lateral shoots and apical shoots of P. amboinicus were inoculated on Murashige and Skoog (MS) culture medium supplemented with different concentrations of BAP, NAA and KIN. When the effect of each growth regulator was considered singly, the highest rate of shoot induction (80% of explants producing shoots) and highest number of shoots produced (2.4 shoots per explant) were obtained from lateral shoot explants cultured on MS media supplemented with 3.0 mg/L BAP within 6 - 7 weeks. Better results were obtained using MS medium supplemented with 1 mg/L BAP + 5 mg/L NAA. Shoot proliferation rose to 85%, while 5.7 shoots per explants were recorded. Among the different media tested for rooting, MS medium supplemented with 1.0 mg/L IBA was the most effective for root induction. The quality of the roots obtained was better than that obtained using MS media supplemented with NAA or IAA.

Micropropagation of Kebericho: An Endandered Ethiopian Medicinal Plant

Echinops kebericho, endemic to Ethiopia, is a critically endangered medicinal plant. It is among the most important medicinal plants of the country, valued primarily for its root parts. The commercial harvesting and sale of roots of E. kebericho have threatened local populations. This study aimed to develop micropropagation protocol for E. kebericho using shoot tip explants. The study started with seed germination test using seeds stored for different months. Shoot tips from in vitro germinated seedlings were cultured on shoot initiation MS media supplemented with 0, 0.1, 0.5, 1.0, 1.5 and 2.0 mg/l BAP or KN alone. Explants were cultured on shoot proliferation media fortified with Kinetin, BAP, and TDZ each at 0.0, 0.5, 1.0, 2.5, 3.0 and 5.0 mg/l either alone or in combination with 0.0, 0.1, 0.25 and 0.5 mg/l NAA. For rooting, full, half and 1/3 strength MS media supplemented with IBA and NAA alone each at 0, 0.05, 0.1, 0.5, 1.0, 1.5 and 2.5 mg/l were used. Growth regulator free MS medium was used as control. Study results showed that 100% germination was recorded in fresh seeds and dropped as low as 65.18% and 22.3% for 3 and 5 months seeds respectively. 1.0 mg/l KN and 0.5 mg/l KN + 0.1 mg/l NAA showed maximum shoot proliferation on shoot induction media and shoot multiplication media respectively. Best rooting was obtained on 1/3 MS containing 1.5 mg/l NAA with 8.23 roots and 4.82 cm root length and established under greenhouse with 83% survival.

Micropropagation of Daylily(Hemerocallis fulva)from Crown-Tip Explants and Assessment of Somaclonal Variation of in Vitro-Propagated Plants Using SCoT Markers

Determination of the somaclonal variation of in vitro-propagated plants is crucial to determine the appropriate micropropagation protocol and growth regulators for commercial scale multiplication.In this research,nine multiplication media(MM)augmented with different concentrations of 6-benzyl adenine(BA),Kinetin(Kin),and Thidiazuron(TDZ),Three rooting media(RM)supplemented with three levels ofα-naphthalene acetic acid(NAA)and three types of soil mixtures(v/v);Coco peat/Vermiculite/Sand(CVS),Peat moss/Perlite/Sand(PPS)and Peat moss/Perlite(PP)were used in the micropropagation protocol of daylily plants.MM2 showed the maximum shoot length and the number of leaves,while MM9 showed the maximum number of shoots.The RM1 showed the maximum root length and the number of roots.During acclimatization,CVS,PPS,and PP soil mixture showed similar performance except the CVS mixture showed lower performance regarding plant height and diameter.The genetic fidelity of micropropagated plants was evaluated using Start Codon Targeted(SCoT)Markers.Six SCoT primers amplified 51 scorable bands with an approximate range from 146 bp to 1598 bp size.Thirty one out of 51 loci were presented in the mother plants.40 loci were polymorphic,11 were monomorphic and 7 were unique.The amplification patterns of the micropropagated plants demonstrated genetic integrity to the mother plant ranging from 84.32 to 47.06 and somaclonal variations ranging from 52.94 with 5 mg/l BA pathway to 15.68 with 1mg/l TDZ pathway,thus demonstrating that the homogeneity and the variation of the micropropagated plants affected by the type and the quantity of the plant growth regulator used during multiplication subcultures.This research can be successfully used for other ornamental and medicinal plants’bulk multiplication,germplasm conservation,and future genetic improvement.

Red sandalwood(Pterocarpus santalinus L. f.): biology,importance, propagation and micropropagation

Pterocarpus santalinus L. f.(Fabaceae;red sanders) is prized for its wood whose colour and fragrance is due to the presence of santalins that have pharmaceutical and industrial uses. Red sanders is listed as an endangered plant species on the IUCN red data list as a result of the exploitation of its wood and essential oil. This review emphasizes the pollination biology, seed germination, vegetative propagation and micropropagation of P. santalinus. Excessive use of P. santalinus has also caused the emergence of various adulterants, so accurate identification is essential.

In Vitro Micropropagation of Himalayan Weeping Bamboo, Drepanostachyum falcatum

Plant growth hormone BAP (benzyl amino purine), KIN (kinetin), NAA (1-naphthalene acetic acid) and IBA (indole-3 butyric acid) effect was studied on in vitro multiplication of shoots and rooting of Drepanostachyum falcatum. In vitro micropropagation of himalayan weeping bamboo is explained by in vitro shoot induction and proliferation. Excised explant with axillary bud is surface sterilized with 0.1% HgCl2 for 10 - 12 minutes, cleaned with 90% ethanol and inoculated on liquid Murashige and Skoog (MS) culture medium supplemented with different concentrations of BAP/ KIN. Effect of BAP/KIN on shoot induction is with different rate and number of shoots produced by explants with axillary bud cultured on MS media supplemented with 0.0 mg/L BAP/KIN - 5.5 mg/L BAP/KIN. Shoot multiplication with highest rate is achieved on MS medium supplemented with 3.5 mg/L BAP after 4th sub-culturing. The most effective with highest rate and number of root induction combination is 6.5 mg/L IBA after 5 weeks. The roots produced by 6.5 mg/L IBA is best compared with other combination of auxin NAA (1-naphthalene acetic acid).

Influence of BA and IBA or NAA Combinations on Micropropagation of Cryptolepis sanguinolenta

The study being the first of its kind established an efficient protocol for micropropagation of Cryptolepis sanguinolenta, an important endangered medicinal plant species, used in the treatment of Malaria. For shoot induction, semi hard wood nodal segments were maintained on MS (Murashige and Skoog) nutrient medium supplemented with MS vitamins, 30 g/L sucrose, 3% gelrite and various auxin and cytokinin combinations. Treatments involved 6-benzyladenine (BA) at 0.5, 1.0, 1.5, 2.0, 2.5 or 3.0 mg/L in combination with 0.1 mg/L Indole 3-butyric acid (IBA) or Naphthaleneacetic acid (NAA). Control consisted of hormone free MS medium. BA and IBA combinations were found to be more efficient in shoot regeneration than the BA and NAA combinations. Cultures maintained on MS medium supplemented with 3 mg/L BA, in combination with 0.1 mg/L IBA recorded the highest shoot induction (100%), mean shoot length (1.28 cm) and mean number of nodes per explant (2.5). This, however, did not differ significantly from cultures maintained on 2 or 2.5 mg/L BA and 0.1 mg/L IBA supplemented MS medium. Regenerated shoots were transferred onto different media for root induction. Treatments consisted of full strength MS medium augmented with either 0.5 or 0.1 mg/L IBA, hormone free half strength MS medium and half strength MS medium augmented with 0.01 mg/L IBA. After six weeks of culture, no rooting was recorded in all treatments with the exception of half strength MS medium supplemented with 0.01 mg/L IBA, which recorded 60% rooting. Regenerated plantlets were successfully weaned and established in the greenhouse.

Leaf Responses of Micropropagated Apple Plants to Water Stress:Changes in Endogenous Hormones and Their Influence on Carbohydrate Metabolism

The changes in the concentrations of endogenous hormones and their influence on carbohydrate metabolism in leaves of micropropagated Fuji apple plants were studied under water deficiency stress. The results showed that water stress induced a rapid increase in the concentration of abscisic acid （ABA） and led to a decrease in concentrations of both zeatin and gibberellins （GAs）. The concentration of indole-3-acetic acid （IAA） changed in an independent manner, which was not correlated with the different levels of water stress. With regard to the carbohydrates, the contents of sorbitol and sucrose increased, whereas the content of starch decreased. The increase in the concentration of ABA was significantly correlated with both the increase in the activity of aldose-6-phosphate reductase （A6PR） and the decrease in the activity of sorbitol dehydrogenase （SDH）, indicating that ABA played a regulatory role in sorbitol metabolism. The concentration of ABA was positively correlated to the activity of sucrose-phosphate synthase （SPS） but negatively correlated to the activities of acid invertase （AI） and ADP-glucose-pyrophosphorylase （ADPGppase） in water-stressed plants, which indicated that ABA promoted sucrose synthesis and inhibited sucrose degradation and starch synthesis at the same time. Under conditions of water stress, the decrease in the level of zeatin was accompanied by a decrease in the activities of SDH and ADPGPPase. GAs concentration showed positive correlation with ADPGPPase activity. IAA showed no significant correlation with any of the enzymes tested in this study. The results of this study suggested that ABA might be one of the key factors regulating the distribution of carbohydrates under water stress. The metabolism of sorbitol and starch under conditions of water stress might be regulated by the combined action of many plant hormones.

A possibility of saving an endangered endemic of the Lake Baikal shore, <i>Hedysarum zundukii</i>Peschkova (<i>Fabaceae</i>Lindl.) using clonal micropropagation

Hedysarum zundukii Peschkova is one of the Fabaceae endemics of the flora of the Lake Baikal west shore. Because of its very poor renewal by seed production and seedling appearance biotechnological method, clonal micropropagation has been elaborated in order to improve its chances of conservation. A protocol for clonal micropropagation, including introduction, propagation, rooting, acclimatization, field cultivation and prolonged cold storage has been elaborated. Half-dose MS salts, benzylaminopurine 1 mg/dm3 and 2% sucrose were optimal components of the medium for clonal micropropagation. Sucrose was the superior carbon source by comparison with glucose and maltose. It was found that some agar brands were better for propagation, whereas other ones were better for rooting. Transplants produced from acclimatized plantlets vegetated successfully in field conditions, but did not survived in Irkutsk after wintering. However, the same transplants planted into their natural population survived successfully. Micropropagated plantlets retained their ability for propagation in vitro after 10 -12 months of cold storage at 4℃ with illumination. It was concluded that clonal micropropagation may be used as an additional means for conservation of H. zundukii.

The Biomass Dosage Influences the Effects of Diethyl Aminoethyl Hexanoate on Micropropagation of Echinacea purpurea (L.) Moench

The plant growth regulator diethyl aminoethyl hexanoate (DA-6) has proved highly effective on micropropagation of the medicinal plant purple coneflower (Echinacea purpurea (L.) Moench), however, sharp variation of the effects existed among explants in the same treatment, making the application of DA-6 in micropropagation difficult. In order to clarify factors that influencing the treating results of DA-6, explants with different biomass dosage were prepared and inoculated onto medium supplemented with different concentrations of DA-6. It was found that among the three kinds of biomass dosage explants, the lowest biomass explants required the lowest concentration of DA-6, and the highest biomass explants required the highest concentration of DA-6 for the best results on adventitious buds regeneration. Similar results were obtained when regenerated buds of three different biomass dosages were cultured. It could be concluded from the above experimental results that for achieving better DA-6 application results, the concentration of DA-6 should be determined not only by the types but also by the biomass dosage of the explants. The present finding might help to improve the micropropagation efficiency in E. purpurea, and might be applicable for other species

Comparative micromorphological study of wild and micropropagated Dioscorea bulbifera Linn

Objective:To study the leaf epidermis of wild and micropropagated Dioscorea bulbifera Linn.(D.bulbifera)in order to document useful diagnostic features that may be employed for correct crude drug identification and to clear any taxonomic uncertainties in the micropropagated medicinal plant.Methods:Growth responses of micropropagated D.bulbifera were observed on Murashige Skoog medium supplemented with 6-benzylamino purine(1.0 mg/L)+α-naphthaleneacetic acid(0.2 mg/L)+cysteine(20 mg/L)using nodal segments as explants.Leaves of the wild and micropropagated plants were studied microscopically.Results:More than 80%shoot regeneration and formation of 10%-30%whitish-brown callus were observed within 3 weeks.The highest root proliferation was obtained from Murashige Skoog medium of 6-benzylamino purine(0.05 mg/L)andα-naphthaleneacetic acid(0.01 mg/L)with mean root length of(27.00±1.25)mm and elongated single shoot of mean length(38.00±11.09)mm.Leaf epidermal features that revealed similarities between the wild and micropropagated plants included amphistomatic condition,presence of mucilage,glandular unicellular trichome with multicellular head,polygonal cells with smooth walls,stomata type and shape.Slight variations included thick cuticular wall with closed stomata in wild plant compared to thin walled opened stomata in the in vitro plant.Opening of stomata accounted for larger average stomata sizes of(7.68±0.38)μm and(6.14±0.46)μm on the adaxial and abaxial surfaces,respectively of the micropropagated plant compared to the wild.Conclusions:The diagnostic features obtained in the study could serve as a basis for proper identification for quality control for standardization of the medicinal plant.

Influencing in vitro clonal propagation of Chonemorpha fragrans(moon) Alston by culture media strength,plant growth regulators,carbon source and photo periodic incubation

Chonemorpha fragrans is an endangered medicinal woody climber,regarded among alternative plant sources of camptothecin.Camptothecin is a monoterpene indole anti-cancer alkaloid with annual trade value of over three billion U.S.dollars in the recent,and is used in the production of its analog drugs approved for the chemotherapy of cancer of varied types.Effects of plant growth regulators,culture media strength and photoperi-odic duration on the micropropagation ef ciency of C.fragrans from nodal segment explants were studied on Murashige and Skoog(MS)medium amended with Thidiazuron(TDZ),Benzylaminopurine(BAP)or Kinetin(Kin).Thidiazuron was more ef cient over BAP and Kin when half basal MS medium was used over full or quarter strength.Results of carbon source experiment showed sucrose as the most effective over glucose,fructose,and maltose in the clonal production.Studies on the photope-riodic incubation duration showed 12 h as the best light period and sub or supra-optimal resulted in the production of abnormal and albino micro shoots.Experimental results on the evaluation of physiological,biochemical parameters showed the role of pigment molecules and antioxidant systems in the production of albino micro shoots.

Effects of Growth Regulators on Biomass and the Production of Secondary Metabolites in Peppermint (<i>Mentha pi-perita</i>) Micropropagated <i>in Vitro</i>

The effects of plant growth regulators on peppermint (Mentha piperita) cultured in vitro were studied for the purpose of maximizing growth and essential oil production in micropropagated plants. The basal medium was experimentally supplemented with the auxin 4-indol-3-ylbutyric acid (IBA) and the cytokinin 6-benzylaminopurine (BAP) individually and in combination. Supplementation with BAP alone resulted in the highest values for root length, root dry weight, shoot length, and numbers of nodes, leaves, and ramifications. Treatment with IBA alone or with IBA + BAP resulted in a ~50% increase in shoot fresh weight. The production of secondary metabolites was affected only by the addition of cytokinin, which resulted in a ~40% increase in the total yield of essential oils (EOs). Similar trends were observed for yields of the major EO components (menthone, menthol, pulegone, and menthofuran). Our findings demonstrate that the application of growth regulators increases EO production and biomass concomitantly in an herbaceous species rich in commercially valuable terpenes.

Generation of Virus Free Potato Plantlets through Meristem Culture and Their Field Evaluation

Different aspects of micropropagation through meristem culture for the production of virus indexed source plants, in vitro tuberization and field evaluation of the in vitro regenerated plants were studied on four commercial cultivars of potato (Solanum tuberosum L.) viz., Diamant, Cardinal, Shilbilati and Lalpakri. The investigation was conducted at Rajshahi, Bangladesh from December 2010 to March 2012 to produce virus-free potato plantlets through meristem culture, shoot multiplications with root induction as well as their acclimatization and evaluation of morphological characters and tuber yield under field condition. Shoot tips of 25 - 30 day old field-grown plants of above mentioned four cultivars were used for meristem isolation. After isolation, meristems of these varieties of potato were cultured on “M” shaped filter paper bridge in Murashige and Skoog (MS) liquid medium. Four different treatments of media formulations viz. 0.1 mg/L KIN + 0.1 mg/L GA3, 0.1 mg/L KIN + 0.5 mg/L GA3, 0.5 mg/L KIN + 0.1 mg/L GA3 and 0.5 mg/L KIN + 0.5 mg/L GA3 were used as plant growth regulators. From these formulations MS + 0.1 mg/L KIN + 0.5 mg/L GA3 was found to be the best for the primary establishment of meristem culture. The primarily established meristems were subcultured on to MS semisolid basal medium supplemented with four different treatment combinations of hormones viz. 0.5 mg/L BA + 1.0 mg/L IBA;0.1 mg/L KIN + 0.1 mg/L GA3;0.5 mg/L BA + 0.5 mg/L GA3 and 0.5 mg/L KIN + 0.5 mg/L GA3 were used to identify the suitable media compositions for shoot proliferation. Results showed that out of these four media treatments the formulation 0.5 mg/L BA + 0.5 mg/L GA3 was found to be the best suitable for shoot generation. Among the four cultivars of potato higher frequency of shoot proliferation (number of shoots/explant and longest shoot length) was observed in Diamant, though the highest shoot formation (76%) was recorded in Cardinal. Virus free in vitro grown potato plantlets were obtained through DAS-ELISA test and used substantially for micro-propagation. After gradual acclimatization of rooted plantlets of four potato cultivars, they were transferred into the field for cultivation and established successfully. It was observed from the field study of in vitro meristem-derived plantlets that there were no virus-affected plants. The virus-free exotic varieties were much superior in all vegetative attributes and yield compared to those of indigenous varieties with producing potato plants of normal height. In contrast, the indigenous varieties took a longer time to tuber initiation and maturity, lower plant height and number of leaves per plant, a higher number of tubers but a lower amount of tuber weight per plant, and poorer tuber grade than the exotic varieties. However, the variety Cardinal exposed the best performances in the context of survival percentage of plantlets (90%), days to tuber initiation (DTI), a number of leaves per plant (NL), tuber weight per plant (343.40%) and the percentage of rich tuber grade.

厚荚相思组织培养与快速繁殖

以5年生厚荚相思(Acaciacrassicarpa)的萌芽茎段为外植体进行了组织培养的初步研究.结果表明:改良的MS+6 BA2.0mg/L+NAA0.2mg/L培养基能够较好地诱导芽的分化和增殖;适宜的继代增殖培养基为改良MS+6 BA1.5mg/L+NAA0.2mg/L;较为理想的生根培养基为1/2MS+IBA0.5mg/L,生根率达95.0%;生根苗的移栽基质以黄心土的移栽效果最好,移栽成活率达90.0%.

紫穗槐的离体快速繁殖(英文)

以子叶节为外植体 ,建立起了紫穗槐的快速离体再生系统。经过四周的培养 ,在附加8mg· L- 16 - BA的 MS培养基上能够获得再生频率为 1 0 0 % ,平均每个外植体 5 .2 1个芽点的高效再生植株。以再生植株的茎节为外植体所进行的继代能够在相同的培养基上连续的产生新的不定芽 ,但芽点数要少于起始培养。经过 3周的培养 ,有 82 .5 3 %切下的再生茎段能够在含 2 .0 mg· L- 1IAA的 MS培养基上生根。在所有进行分析过的再生植株中 ,它们的染色体数目都没有发生变异 (2 n=40 )。经过练苗以后 ,再生植株成功地定植于土壤当中并展示了一致的外部形态和生长特性。

濒危黄杜鹃的离体快速繁殖体系研究

以濒危野生黄杜鹃(Rhododendron molleG.Don)茎尖为试材,比较研究3种不同基本培养基和9个激素浓度组合对不定芽增殖的影响,以建立黄杜鹃的快速繁殖体系。结果表明:3种不同基本培养基对野生黄杜鹃不定芽增殖的效果差异显著;相同激素种类与浓度下,WPM培养基上的不定芽增殖系数最大,达10.5。激素种类与浓度对黄杜鹃不定芽的增殖影响差异很大,以单独使用1.0 mg/L ZT最佳,再生率达100%,不定芽增殖系数为10.5。综合分析显示,黄杜鹃不定芽增殖的最佳培养条件为WPM+ZT 1.00 mg/L;健壮、整齐一致的不定芽在1/2 WPM+0.05 mg/L NAA上生根,40 d后生根率可达86.7%,驯化成活率达92.86%。

植物组织培养生物反应器技术研究进展

从植物大规模组织培养的特点、反应器类型和反应器中微环境等方面介绍了生物反应器技术在药用植物微繁殖和天然产物细胞生产中的研究进展。

蓝靛果忍冬茎段离体培养与植株再生

以蓝靛果忍冬的幼嫩茎段为外植体,采用正交设计及方差分析对影响蓝靛果忍冬植株离体培养与植株再生的因素进行了优化研究,培育出了蓝靛果忍冬的组培苗。结果表明:初代培养的适宜培养方案是MS+6-BA1.0mg/L+IBA0.2mg/L,芽分化率达到100%;继代增殖培养的适宜培养方案是MS+6-BA0.5mg/L+IBA0.3mg/L+KT1.5mg/L,增殖系数达到8.7;生根培养的适宜培养方案是1/4MS+IBA1.5mg/L,生根率达到96.7%。生根试管苗在驯化、移栽后成活率可达97.8%。6-BA、IBA和KT配合使用,可以增加蓝靛果忍冬继代培养的丛生芽数量。该试验培育的组培苗的各项指标已达到快速繁育苗木的要求,其技术措施可直接应用于规模化生产。

丛枝菌根真菌对芋组织培养苗生长的影响

于温室盆栽条件下研究了丛枝菌根 (AM)真菌Gigasporarosea、Glomusmosseae和Glomusversi forme对芋 (Colocasiaesculenta)组织培养苗移栽成活率、矿质营养、光合速率及生长的影响。结果表明 ,接种AM真菌能提高芋组织培养幼苗移栽成活率和叶片光合速率 ,降低气孔阻力 ;其叶片和根内氮、磷、钾含量和生长量显著高于不接种对照。认为接种有效AM真菌是促进组织培养苗健康生长的重要技术。