A New Micropropagation Technology of Tilia amurensis:In VitroMicropropagation of Mature Zygotic Embryos and the Establishment of a PlantRegeneration System

Tilia amurensis is an economically valuable broadleaf tree species in Northeast China.The production of highqualityT.amurensis varieties at commercial scales has been greatly limited by the low germination rates.Thereis thus a pressing need to develop an organogenesis protocol for in vitro propagation of T.amurensis to alleviate ashortage of high-quality T.amurensis seedlings.Here,we established a rapid in vitro propagation system forT.amurensis from mature zygotic embryos and analyzed the effects of plant growth regulators and culture mediain different stages.We found that Woody plant medium(WPM)was the optimal primary culture medium formature zygotic embryos.The highest callus induction percentage(68.76%)and number of axillary buds induced(3.2)were obtained in WPM+0.89μmol/L 6-benzyladenine(6-BA)+0.46μmol/L kinetin(KT)+0.25μmol/Lindole-3-butryic acid(IBA)+1.44μmol/L gibberellin A_(3)(GA_(3)).The multiple shoot bud development achievedthe highest percentage(83.32%)in the Murashige and Skoog(MS)+2.22μmol/L 6-BA+0.25μmol/L IBA+1.44μmol/L GA_(3).The rooting percentage(96.70%)was highest in 1/2 MS medium+1.48μmol/L IBA.Thesurvival percentage of transplanting plantlets was 82.22%in soil:vermiculite:perlite(5:3:1).Our study is the firstto establish an effective organogenesis protocol for T.amurensis using mature zygotic embryos.

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.

Efficient Micropropagation of Chestnut Hybrids(Castanea spp.)Using Modified Woody Plant Medium and Zeatin Riboside

The chestnuts genus(Castanea spp.)is comprised of economically important trees native to the Northern hemisphere that are used as food and hardwood timber.Here,a very efficient method for micropropagation of European×Japanese chestnut hybrids(Castanea sativa×C.crenata)is described.Woody Plant Medium was used as the basal medium.In vitro shoots of four rootstock cultivars were micropropagated without shoot-tip necrosis on multiplication medium containing 5.7 or 11.4μmol·L^(−1)zeatin riboside,and were rooted on rooting medium containing 2.46μmol·L^(−1)indolebutyric acid.Monthly shootmultiplication rates for each cultivarwere 2–5 folds.In vitro rooting percentages for four cultivars were 87%for‘Maraval’,67%for‘Marigoule’,93%for‘Marsol’,and 97%for‘Précoce Migoule’.Within a 5 week period,80%–95%of rooted shoots were successfully acclimated under high humidity conditions after they were planted in either soil or rockwool.

Micropropagation of Pinus densiflora and the evaluation of nematode resistance of regenerated microshoots in vitro

To accelerate the breeding and selection of Pinus densiflora Siebold and Zucc. resistance to pine wilt disease, a micropropagation system was established and nematode resistance evaluated in vitro. Cotyledon-hypocotyl explants from 28-day-old seedlings were first cultured on Gresshoff and Doy medium supplemented with 4.0 mg L^(-1) 6-benzyladenine and 0.2 mg L^(-1) a-naphthaleneacetic acid(NAA) to stimulate the formation of buds. Induced buds were subsequently subcultured on Gupta and Durzan medium supplemented with 0.1%(w/v)activated charcoal for elongation. Stem sections derived from shoots were used as explants for the further multiplication. Roots were formed from shoots transferred to woody plant medium containing 0.2 mg L^(-1) NAA for4 weeks. The nematode resistance test showed that symptoms in micropropagated shoots after infection with pine wood nematode(PWN) were similar to those in plants infected in the field. The wilting rate varied from 20 to100% among different clones 18 days after inoculation.The most susceptible clone was Clone 6-4 with a 100%wilting rate, while Clone 8-4 showed a relatively high resistance with a 20% wilting rate. The number of nematodes recovered from Clone 8-4 shoots was significantly lower(P = 0.05) than from Clones 5-10 and 16-4. This work contributes to the breeding of PWN resistance in P.densiflora.

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.

Micropropagation of the Moroccan Endemic Plant <i>Thymus broussonetii</i>Boiss. with Aromatic-Medicinal Value and Conservation Concern

Micropropagation from shoot tips and nodal segments was carried out for the conservation and domestication of spontaneous Moroccan thyme, Thymus broussonetii Boiss. subsp. broussonetii (endemic threatened). The mineral composition of the culture medium, as well as the succession of different growth regulators, influenced the in vitro growth of this species. Sterilized achenes of T. broussonetii were able to germinate on an agar medium containing Gautheret macronutrients with a rate of 25% and a degree of contamination of less than 4%. Shoot apices of 15-day seedlings (two cotyledon leaves) were cultivated on SD + 0.46 μM Kin medium and the explants obtained were transplanted every month. Six macronutrients (MS, B5, SH, SD, MSm and N30K) were tested and N30K was chosen for the following experiments. Seven cytokinins (Kin, BAP, 2iP, DPU, adenine, Zeat and TDZ) at 0.46, 0.93 and 2.32 μM/l were evaluated and the addition of 0.93 μM adenine to N30K medium favored significantly the induction of buds and the elongation of explants. Three polyamines (putrescine, spermidine and spermine) at 2, 5, 10 and 20 μM/l were tested. A better multiplication of buds, shoots and roots was noted for N30K + 10 μM spermine. Cytokinin-auxin combinations led to better root multiplication and an increase in the number of buds and the length of explants, particularly for 0.46 μM Kin + 2.85 μM IAA. Acclimatization was successfully carried out using vitroplants developing a good root system. One month after the start of acclimatization, 97% of T. broussonetii plantlets were healthy. Three months later, they were transplanted into larger pots. 100% of the acclimatized plants developed flowers in the 2nd year between June and August. Re-initiation of the in vitro culture was carried out from sterilized twig segments collected from the acclimatized plants of T. broussonetii with 1 - 2 nodes on the medium N30K + 0.46 μM Kin, and 52.1% of the explants healthily proliferated. Finally, two micropropagation prototypes were developed: shoot tip culture from seedlings obtained after germination of achenes and node culture from acclimatized plants.

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.

An Approach for Micropropagation of Blueberry (<i>Vaccinium corymbosum</i>L.) Plants Mediated by Temporary Immersion Bioreactors (TIBs)

A new procedure for blueberry (Vaccinium corymbosum L.) micropropagation in programmed Temporary Immersion Bioreactors (TIBs based on two separate bottles) was developed for the commercial genotypes Biloxi, Sharp Blue and Brillita. Plant cultures were developed in a controlled environment with 0.4 MPa CO2 enrichment, sucrose-reduced medium, and light intensity of 60 mM m-2·s-1. Principal component analysis showed that component 1 (C1) grouped 64.08% of the total variability, while the first two components accounted for 86.97%. Representation of the principal components demonstrated three clusters corresponding with the blueberry genotypes, and within each cluster plants micropropagated in agar-base medium grouped separately from those plants multiplied in TIBs. Both plant number and total internodes traits (related to the productive efficiency) were demonstrated superior in blueberries propagated in TIBs. Additionally, when transferred to greenhouse conditions, blueberries propagated in TIBs showed higher adaptability and growing rates than those cultured by the conventional approach, altogether evidencing the occurrence of a photomixotrophic stage in the vitroplantlets cultured in TIBs.

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 and Acclimatization of Common Oregano (Origanum vulgare L. Subsp. vulgare) by Shoot Tip Culture

Origanum vulgare L. is a commercially valued species with remarkable biological properties. It is subject to over-exploitation practices that seriously threaten its sustainability for future generations. Thus, micropropagation serves as a tool for the protection and domestication of this species. In this study, we established an in vitro vegetative propagation protocol for Origanum vulgare. This is done through the axillary bud technique by carrying out various tests. Six culture media (MS, MSm, N30K, SD, SH and B5) were tested. Therefore, SD was chosen for the following experiments. Seven cytokinins (adenine (Ad), N6-(2-isopentenyl) (2ip), zeatin (Zeat), kinetin (Kin), benzyladenine (BAP), 1,3-diphenylurea (DPU) and thidiazuron (TDZ) at 5 concentrations (0.44, 1.33, 2.22, 3.11 and 4.44 μM/L) were evaluated. Thus, Kin at 3.11 μM allowed high regeneration of vitroplants, optimal elongation, total rooting of explants, maximum bud multiplication, and absence of hyperhydric explants. In fact, the integration of auxins (indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), and 1-naphthaleneacetic acid (NAA)) into the culture medium and their combinations with 3.11 μM Kinetin contributed to the optimization of the root part. Thus, it was improved in particular in the case of 3.11 μM Kin and 6.27 μM IBA. Three polyamines (Putrescine, Spermidine and Spermine) at different concentrations (1.134, 3.402, 5.67, 7.938 and 11.34 μM/L) combined at 3.11 μM Kin and 6.27 μM IBA were tested. In fact, 1.304 μM putrescine was considered to be the most suitable for in vitro culture of explants, since it allowed optimal propagation of buds and roots, also a high rate of regeneration and rhizogenesis. GA3 at 1.15 μM combined with 3.11 μM Kin and 6.27 μM IBA permitted maximum bud multiplication. The acclimatization was carried out successfully using vitroplants showing good foliar and root development. Thus, three months after acclimatization, the seedlings were transferred into large pots under natural light and temperature conditions. Almost all acclimatized plants developed flowers in the first year between May and July.

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.

Micropropagation of Taxodium hybrid(T.distichum×T.mucronatum)

This study was carried out to optimize the culture media for the micropropagation of Taxodium hybrid Zhongshanshan（ T. distichum × T. mucronatum）.Using the tender stems of Zhongshanshan 301 as the explants,the effects of NAA,6-BA,IBA and KT on the induction of differentiation,proliferation and rooting were evaluated on MS or 1/2 MS medium. The results showed that Zhongshanshan can be proliferated via tissue culture. The combined use of NAA and 6-BA in MS medium greatly promoted the differentiation of Zhongshanshan explants,and the optimal medium was MS ＋ 0. 2 mg/L NAA ＋ 0. 4 mg/L6-BA. The optimal medium for the proliferation of differentiated buds was MS ＋ 0. 3 mg/L NAA ＋ 0. 4 mg/L 6-BA,on which the proliferation rate was up to 3. 8. 1/2 MS medium was more conducive than MS medium to the induction of rooting. The optimal medium for the rooting of tissue-cultured Zhongshanshan shoots was 1/2 MS ＋ 0. 3 mg/L IBA ＋ 0. 2 mg/L NAA.

Micropropagation protocol for Salvadora oleoides

Salvadora oleoides Decne. is a pharmaceutically important plant. Owing to poor seed formation, viability and, germination, and to anthropogenic disturbances, this species is on the verge of extinction. A reproducible micropropagation protocol to increase the population through tissue culture has been standardized and the results are reported here. Callus tissues were initiated from young leaves and stem explants. Leaf calluses proliferated with 1.5 mg/L BAP and 0.9 mg/L 2, 4-D with additives and continuous slow proliferation up to 15 weeks on 0.5 mg/L BAP and additives with 200 mg/L activated charcoal.Direct shoot initiation took place from stem node explants after 12 days; 4–5 shoots per node were produced in 30 days. Shoot clumps elongated and grew further on MS media supplemented with 2 mg/L BAP, 0.2 mg/L NAA and additives, which generated 20–23 shoots. The elongated shoots induced tap roots with 4 mg/L NAA and200 mg/L activated charcoal in 12 days. In vitro raised plants produced secondary roots when transferred to pots containing vermiculite maintained at 28–35 ℃. The plantlets successfully acclimatised in pots containing soil in natural conditions.

Micropropagation of Avocado (<i>Persea Americana</i>Mill.)

Avocado is a high demand, high value tropical fruit recognised for its nutritional value. Being planted as a grafted tree, propagation of avocado refers to propagation of rootstock cultivar, then graft it with bud-wood from a mature scion cultivar. Elite cultivar propagation is critical to maintain the quality of fruit and farm management practices. Avocado propagation through seeds exhibit high genetic variation, hence less appealing for orchard plantings. Rooting of cuttings is only possible through a complex, lengthy and expensive process called “Frolich and Platt method”. This creates limitations on rapid industry expansion due to scarcity and high price of plants in many countries. Alternative propagation methods are sought over 5 decades. Potential of micropropagation has been well demonstrated for wide variety of economically important plants. Commercial application of micropropagation for avocado will undoubtedly boost the industry around the globe. In this review, we present the developments in micropropagation of recalcitrant species, avocado, over the last 45 years. We summarise the culture media composition, hormones, growth conditions for different stages of avocado micropropagation pipeline, elaborating on cultivar specificity for in vitro success, and problems encountered under in vitro conditions and during acclimatisation. Overview of the current knowledge is critical to focus on important aspects in protocol optimisation, to develop an efficient and effective micropropagation system for avocado as well as other woody plant species recalcitrant for micropropagation.

Rooting of Stem Cuttings with Different Indole 3 Butyric Acid (IBA) Treatments and Development of Micropropagation Protocol for <i>Piper betle</i>L. Node Culture

The present study, conducted during 2016 and 2017 seasons, aimed to investigate the effect of IBA on rooting of Piper betle L. stem cuttings (softwood and semi-hardwood). The experiment was undertaken in misting house field 2 UPM using the sand media to determine the adventitious roots initiation and development using the histological method. The cuttings were treated with different IBA concentrations (0, 500, 1000, 1500 and 2000 mg/L). The nodes explants were used in the development of a protocol for in vitro propagation of P. betle L., with different concentrations of Clorox with different times of immersion (20% Clorox 10 minutes, 30% Clorox 10 minutes, 20% Clorox 20 minutes, and 30% 20 minutes). In multiplication of the plantlets, Murashige and Skoog (MS) medium with different concentrations of BAP (0, 0.5, 1.0, 2.0 mg/L) were used to investigate the rooting of the explants. The results indicated that the types of the cuttings were different in the rooting capacity and the length of the roots. Moreover, it was found that in comparison with the control treatment, by a rise in the concentrations of the IBA, there was a significant upsurge in the rooting percentage, the root diameter, and the number of the roots. The results indicated that the types of cutting with 1000, 1500 and 2000 mg/L IBA perform better in the root percentage (100%) in the semi hardwood cuttings. The best results, however, were 2000 mg/L IBA in the semi hardwood cuttings, with the number of the roots to be 35.05, and the fresh weight of the roots to be 3.94 g, the dry weight of the roots to be 0.33 g, the length of the roots to be 391.88 cm, the roots diameter to be 1.21 mm, the surface area of the roots to be 121.83 cm2, and the root volume to be 2.99 cm3. Nonetheless, the optimal concentration of Clorox with the time immersion was 20% with the 20-minute immersion time, which produced a shoot induction percentage of 30% dead explants and a mean number of 70.00 shoots per explant and the optimal concentration of benzylaminopurine (BAP) at 1.0 mg/L. It is of note that a shoot induction percentage of 22.29% and a mean number of 4.1% number of auxiliary bud per treatment. P. betle shoots in MS medium without PGR MS (0.0) yielded a good rooting.

<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.

Interaction Effect of Plant Growth Regulators on Shoot Micropropagation of Aromatic Plant Origanum elongatum (Bonnet) Emberger &Maire

Origanum elongatum (Bonnet) Emb. & Maire, is a medicinal, aromatic and endemic plant of Morocco, characterized by its pharmacological effects, and is commonly used for the production of essential oils and aromas, resulting in high harvest and overexploitation pressure. This is why the present study aims to implement the in vitro micropropagation of Origanum elongatum for optimal vitroplant production. Six macroelements were tested (SH, SD, N30K, MS, MSm and B5) and the SD medium was selected for vegetative propagation of the explants. Seven cytokinins: adenine (Ad), N6-(2-Isopentenyl) adenine, zeatin (Zeat), kinetin (Kin), benzyladenine (BAP), 1,3-diphenylurea (DPU) and thidiazuron (TDZ) were then evaluated at five concentrations (0.44, 1.33, 2.22, 3.11 and 4.44 μM/L) on growth, development, budding, rooting and hyperhydricity. 0.44 μM Kin was selected and combined with three auxins: indole-3-acetic acid (IAA), indole-3-butyric acid (AIB), and 1-naphthaleneacetic acid (NAA) at four concentrations (1.14, 2.85, 4.56 and 6.27 μM/L) to improve rooting and association with 1.14 μM IAA was shown to be efficient for roots development. Different concentrations of gibberellic acid (0.29, 1.5, 2.60 and 2.89 μM/L), combined with 0.44 μM/L Kin and 1.14 μM/L IAA, were tested and 2.60 μM/L GA3 gave maximum buds and shoots. Then, the combination of three polyamines at five concentrations (1.134, 3.402, 5.67, 7.938 and 11.34 μM/L) with 0.44 μM Kin and 1.14 μM/L IAA showed an increase in the number of buds and shoots for 7.938 μM/L putrescine and 3.402 μM/L spermine. Finally, seedlings with good foliar and root development were acclimatized.

Influence of Meta-Topolin on Efficient Plant Regeneration via Micropropagation and Organogenesis of Safflower (<i>Carthamus tinctorius</i>L.) cv. NARI-H-15

The effect of meta-Topolin (mT) was assessed to develop a reliable protocol for efficient plant regeneration of safflower (Carthamus tinctorius L.) cv. NARI-H-15. For micropropagation, 7 - 9 days old shoot-tip explants cultured on MS basal medium supplemented with 3.0 mg/L meta-Topolin (mT) + 0.5 mg/L CPPU showed 97.7% adventitious shoot formation (42.4 shootlets) than node after 45 days of culture. For organogenesis, the seedling explants of immature leaf cultured on 1.5 mg/L CPPU or 1.5 mg/L NAA fortified medium produced high amount of callus than cotyledon and stem calli after 60 days of culture. However, MS basal medium fortified with 4.0 mg/L mT + 1.5 mg/L CPPU was found beneficial to stimulate 100% organogenic response (74.7 shootlets) from immature leaf calli than cotyledon and stem derived calli after 45 days of culture. The healthy plantlets obtained from micropropagation and organogenesis process cultured on 1/4 MS basal salts, 1.5% sucrose (w/v) and 0.8% agar (w/v) medium supplemented with NAA (1.5 mg/L) and mT (0.1 mg/L) produced maximum of 96% (12.8 rootlets) and 84% (7.3 rootlets) adventitious rooting, respectively than mT and CPPU tested medium. However, maximum of 67% and 42% survival rate was noticed when in vitro raised plants from micropropagation and organogenesis were hardened in pots containing soil mix and maintained under green house condition. This optimized regeneration protocol might be helpful in regeneration of new genotypes and cultivars of safflower to improve agronomic traits through in vitro selection process and Agrobacterium-mediated genetic transformation system.

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.