Figures (2)  Tables (3)

    • Figure 1. 

      Prototype of gene transfers and plant regeneration in spice crops (CRISPR: Clustered Regularly Interspaced Short Palindromic Repeats; Cas: associated protein; gRNA: guide ribonucleic acid; ds DNA: doubled stranded deoxy ribonucleic acid; PAM: protospacer adjacent motif/short DNA sequence).

    • Figure 2. 

      Integration of gene transfers, plant tissue culture, and speed breeding techniques in spice crops under a controlled environment ecosystem (CRISPR- Clustered Regularly Interspaced Short Palindromic Repeats; Cas9- associated protein 9).

    • S. No. Spices Medicinal importance Country Media + growth regulator levels Tissue culture methods Explants Callus induction, shoot regeneration, and root regeneration
      (d: days, w: weeks)      		 Transformation methods Regeneration and transformation efficiency Ref.
      1 Nigella sativa Reduce blood pressure, cholesterol, triglyceride levels, improve insulin sensitivity and pancreatic beta cell function Iraq Hairy root: MS + 200/300/100 mg/L cefotaxime Hairy Root Culture Seeds NA, NA, 20−25 d Agrobacterium rhizogenes +
      x-gluc + 35s      		 33% of hairy root induction with 4.2 hairy roots/segment; 6.31 mg/g thymoquinone content in hairy roots which was higher compared to seed (4.19 mg/g) and normal seedlings (1.27 mg/g) [80]
      2 Piper nigrum L. Regulate blood pressure, lower cholesterol levels, anticancer potential, neuroprotective effects, promotes fat burning and weight loss, regulates blood sugar levels India Callus: SM media + 1.5% sucrose + 100 μg/L cefotaxime + 25 μg/L kanamycin Direct somatic embryogenesis Mature Seeds 3−5 w, 4 w, NA Agrobacterium mediated + PCR
      + RAPD      		 Average of 9 hardened putative transgenic plantlets per gram of embryonic mass; Dot blot confirmed NPT II gene presence in 62 of 74 putative transformants. Southern analysis confirmed stable NPT II integration in six of 17 screened transformants [81]
      3 India Callus + plant regeneration: MS + kinetin + NAA; shoot: MS + 1 mg/L IAA / 1 mg/L IBA; root: MS + 0.2 mg/L NAA Organogenesis + somatic embryogenesis Shoot tips, nodes, leaf NA, NA, NA − NA [82]
      4 India Sterile water + 5% alginate Synthetic seeds Shoot buds NA, NA, NA − NA [82]
      5 Elethania cardamomum Matra. Blood sugar regulation, neuroprotective effects, metabolic and weight management, aphrodisiac and hormonal balance India MS + 1 mg/L BAP + 0.5 mg/L IBA + 5% alginate Synthetic beeds Callus NA, NA, NA − NA [82]
      6 Cinnamomum verum Persl. Regulation of blood pressure and heart rate India Shoot: MS + 0.5 mg/L NAA / 0.5 mg/L BAP Organogenesis Shoot tips NA, NA, NA − NA [82]
      7 Cinnamomum verum Persl. Regulation of blood pressure and heart rate India MS + 3 mg/L BAP + 0.5 mg/L kinetin Synthetic seeds Shoot buds NA, NA, NA − NA [82]
      8 Syzgium aromaticum L., Merr. & L. M. Perry For digestive health, antinfallmatory effects India Shoot: MS + 0.5 mg/L NAA / 0.5 mg/L BAP Organogenesis Shoot tips NA, NA, NA − NA [82]
      9 Zingiber officinale Roscoe Treating nausea, motion sickness, constipation, and osteoarthritis, etc. India Haploid lines + callus- MS + 2−3 mg/L 2,4-D; plant regeneration: MS + 10 mg/L BA / 0.2 mg/L 2,4-D Anther culture Anthers - uninucleate stage NA, NA, NA − NA [82]
      10 Shoot: MS + 4 mg/L BA Organogenesis Rhizome buds NA, 5 w, NA − NA [82]
      11 MS + 1 mg/L BAP + 5% alginate Synthetic seeds Somatic embryo NA, NA, NA − NA [82]
      12 Myristica fragrans Sedative and sleep aid, antimicrobial properties, cognitive support, digestive health, anti-inflammatory effects, etc India Shoot: WPM + 3 mg/L BAP / 1 mg/L kinetin Organogenesis Shoot tips NA, NA, NA − NA [82]
      13 Curcuma longa L.) Management of osteoarthritis, fever, depression, liver diseases, and high cholesterol India Callus + shoot: MS + 10% coconut milk + 2.5 mg/L BAP Organogenesis Vegetative buds NA, NA, NA − NA [82]
      14 C. longa L. Management of osteoarthritis, fever, depression, liver diseases, and high cholesterol India MS + 1 mg/L BAP + 0.5 mg/L IBA + 5% alginate Synthetic seeds Adventitious buds NA, NA, NA − NA [82]
      15 Vanilla planifolia Jacks. For fever, spasms, blood clotting issues, and gastrointestinal distress India Plant regeneration: MS + 0.5 mg/L IBA Secondary metabolites-MS +
      0.1 mg/L 2,4-D / 0.5 mg/L kinetin / 1 mg/L IBA      		 Somatic embryogenesis, suspension culure Aerial root NA, NA, NA − NA [82]
      16 Vanilla planifolia Jacks. For fever, spasms, blood clotting issues, and gastrointestinal distress India Sterile water + 5% alginate Synthetic beeds Shoot buds NA, NA, NA − NA [82]
      17 Camphor Cinnamomum camphora L. J. Persl., Cinnamomum verum Diagnosis in resuscitation, heat clearance, pain relief, fever, convulsions, stroke, sputum fainting, sputum coma, laryngeal pain, mouth pain, anthrax, bloodshot eye Mauritius Callus: MS + 1 mg/L BAP + 0.005-5 mg/L TDZ; Shoot: MS + 1 mg/L BAP + 2.5 mg/L TDZ Organogenesis Shoot tips, leaf 4 w, 6−8 w, 2 w − C. verum explants: Responded by sprouting. Rooting: 100% shoots rooted within two weeks on basic MS medium No Agrobacterium-mediated transformation was performed, so transformation efficiency is not applicable in this study [83]
      18 Cumin Cuminum cyminum L. Reduce blood pressure, cholesterol, triglyceride levels, improve insulin sensitivity, and pancreatic beta cell function India Callus + shoot: B5 media + 2 μM BA + 0.5 μM NAA Indirect organogenesis Seeds-embryo NA, 29 d, NA Agrobacterium mediated + GFP + RT-PCR; gene gun method + GFP
      + RT-PCR      		 Explants showed highest regeneration and transformation efficiency at 0.5 OD600 with 24 h co-cultivation, compared to other OD and time combinations. [84]
      19 Zingiber officinale Roscoe Treating nausea, motion sickness, constipation, and osteoarthritis, etc. India Callus: B5 media + 2 μM BA + 0.5 μM NAA Organogenesis Seed- embryo NA, NA, NA Agrobacterium mediated + GFP + PCR Gene gun + PCR Regeneration efficiency of transformed explants was highest (96.2%) on 0.4 M sorbitol-containing media, with the lowest browning rate (4.07%) compared to 0.2 and 0.6 M sorbitol. The transformed cumin embryos showed 51.93% regeneration efficiency [84]
      20 Cuminum cyminum L. Reduce blood pressyre, cholesterol, triglyceride levels, improve insulin sensitivity and pancreatic beta cell function Iran Direct somatic embryogenesis: B5 media + 1/10 μM TDZ Indirect somatic embryogenesis: callus: B5 media + 0.1−0.4 mg/L / 1 mg/L BAP + 0.2/0.4 mg/L NAA; shoot: MS + 0.4 mg/L BAP + 0.1 mg/L NAA, MS + 1 mg/L BAP + 0.1 mg/L NAA Direct ir Indirect somatic embryogenesis Mature seeds 14 d, 5 w, NA − Direct regeneration was highest (57.5%) at 10 μM TDZ and lowest (6.67%) at 1 μM, while indirect regeneration peaked (25.83%) with 0.1 mg/L NAA + 0.4 mg/L BAP; Shahdad and Afghanistan accessions showed superior response. [85]
      21 Elethania cardamomum Matra. Blood sugar regulation, neuroprotective effects, metabolic and weight management, aphrodisiac and hormonal balance India Callus: MS + 9 μM 2,4-D / 2−3 μM kinetin − Rhizome 4−8 w, NA, NA Agrobacterium mediated + PCR + southern blot + dot blot No specific transformation or regeneration efficiency data [86]
      22 E. cardamomum Matra. Blood sugar regulation, neuroprotective effects, metabolic and weight management, aphrodisiac and hormonal balance India Shoot: MS + 1 mg/L BAP + 0.2 mg/L kinetin / 0.2 mg/L IAA / 0.1 mg/L calcium panthothenate + 5% cocnut milk Organogenesis Vegetative buds NA, NA, NA − NA [87,88]
      23 Cinnamomum verum Persl. Regulation of blood pressure and heart rate Sri Lanka Shoot: ½MS + 1.5 mg/L BAP + 0.2 mg/L IAA; root: ½MS 0.1 mg/L NAA + 4 mg/L BAP Organogenesis Axillary buds NA, 14 d, 6 w − Stem elongation: 19.5 mm after 14 d; leaf initiation: 2.06 leaves/plantlet after 14 d; root length: 6.7 cm after 6 w (on full MS + 0.1 mg/L NAA + 4.0 mg/L BAP + 1 g/L activated charcoal) [89]
      24 Syzgium aromaticum L., Merr. & L. M. Perry For digestive health, antinfallmatory effects India Shoot: ½MS salts + B5 media + 3 mg/L BAP / 0.5 mg/L NAA Organogenesis Nodes, shoot tips NA, 4−5 w, NA − 6−8 shoots were obtained when 3 mg/L BAP and 0.5 mg/L NAA used in medium. Furthermore, 1−3 mg/L BAP and 0.1 mg/L NAA proved most effective for promotion of shoot proliferation. [90]
      25 Syzgium aromaticum L., Merr. & L. M. Perry For dental health, digestive aid, antimicrobial properties, blood sugar regulation, liver protection, aphrodisiac and reproductive health New Zealand Plant regeneration: white and voisey (CR) media + MS salts + B5 vitamins + 1 mg/L BAP + 1 mg/L NAA; root: white and voisey (CR) media + hormone free − Seeds NA, 4 w, 2−3 w Agrobacterium mediated + GUS
      + PCR      		 NA [91]
      26 Zingiber officinale Roscoe Treating nausea, motion sickness, constipation, and osteoarthritis, etc. India MS + 1 mg/L BAP/ 2 mg/L BAP; MS + 1 mg/L BAP + 0.5 mg/L NAA; MS + 1 mg/L BAP + 1 mg/L NAA; MS + 2 mg/L BAP + 1 mg/L NAA; MS + 3 mg/L BAP + 1 mg/L NAA Direct organogenesis AerialaStem- axillary meristem, apical meristem NA, 2 w, 2 w − 85% of plantlets were successfully hardened and established in the field, with 95% success from basal (axillary meristem) and 70% from middle (apical meristem) stem explants. [92−94]
      27 India Callus: MS + 9−22.6 μM 2,4-D; shoot: 0.9 μM 2,4-D + 44.4 μM BA; root: MS + 5.4 μM NAA Indirect organogenesis Aerial shoot, young leaf 4 w, 4 w, 5 w − Organogenesis and regeneration occurred at 11.9 μM 2,4-D + 44.4 μM BA with 80% soil establishment; transformation efficiency not reported. [95]
      28 Myristica fragrans Sedative and sleep aid, antimicrobial properties, cognitive support, digestive health, anti-inflammatory effects, etc. India Callus: WPM + 1 mg/L BA + 1 mg/L NAA Indirect organogenesis Matured unopened fruits NA, NA, NA − [95]
      29 Z. officinale Roscoe Treating nausea, motion sickness, constipation, and osteoarthritis, etc. India − − − NA, NA, NA Agrobacterium mediated + GUS
      + PCR      		 The regeneration of hygromycin-resistant plantlets was confirmed, it does not include detailed regeneration efficiency [96]
      30 Z. officinale Roscoe Treating nausea, motion sickness, constipation, and osteoarthritis, etc. India Shoot: ½MS + 1−10 mg/L 2,4-D / 3 mg/L BA Organogenesis Shoot buds. leaf discs, pseudostem 2−4 w, NA, NA Agrobacterium mediated + GUS
      + PCR      		 Using young bud as explant, transformation frequency ranged from 1.1% to 2.2%, with slow callus growth under antibiotic selection [97]
      31 Allium sativum L. Management of heart health, immune boosting, digestive health, bone health, aphrodisiac amd reproductive health Korea, Croatia Callus: MS + 2 mg/L 2,4-D; shoot: MS + 0.1 mg/L BAP Indirect organogenesis Shoot tips 4 w, 14 w, NA − Regeneration declined with callus age, but healthy plantlets were obtained from callus up to 16 months old across all clones. [98,99]
      32 Mexico, Turkey Callus: MS + 2.2−4.5 μM 2,4-D, MS + 2.2−4.5 μM 2,4-D + 2.2−4.6 μM kinetin; Shoot: MS + 4.4 μM kinetin Indirect organogenesis, root tip culture Root tips 8 w, 3−6 w, NA − Regeneration data showed a high efficiency—up to 170 plantlets per gram fresh weight (FW) of callus—when cultured on MS medium with 4.6 μM kinetin + 4.5 μM 2,4-D. [100,101]
      33 Crocus sativus L. For cardiovascular health management, reduction in inflammation, and helps manage depression India Callus: MS + 0.5 mg/L 2,4-D + 1 mg/L BAP + 1 mg/L IAA; plant regeneration: 0.5 mg/L TDZ +1 mg/L IAA + 0.1 g/L activated charcoal Indirect somatic embryogenesis Corm slice 3−4 w, 4 w, NA Agrobacterium mediated + PCR
      + southern blot      		 4% transformation efficiency with Agrobacterium, > 70% callus induction, and cormlet regeneration in 8 w using TDZ + IAA + activated charcoal [102]
      34 Japan, India Callus: 2 μM BA + 2 μM NAA + 40, 60 g/L table sugar; root: 8.8 μM IBA + 40 g/L table sugar − Corms 8 w, NA, 12 w − Cormlet regeneration: 70.0 ± 0.30 cormlets per corm slice on ½ MS + TDZ (20 μM) + IAA (10 μM) + sucrose (40 g/L) Cormlet germination: 90% on MS + BAP (20 μM) + NAA (15 μM) Cormlet enlargement: Up to 2.5 g on MS + TDZ (15 μM) + IAA (12.5 μM) + sucrose (30 g/L) Flowering: 25% of 2.5 g cormlets flowered under greenhouse conditions [103−105]
      35 Curcuma longa L., Curcuma mangga valeton & zijp Management of osteoarthritis, fever, depression, liver diseases, and high cholesterol Thailand Embryo: MS + 5 mg/L 2,4-D / 5 mg/L NAA; shoot: MS + 3 mg/L BA + 0.5 mg/L NAA + 3% maltose; root: MS + 3 mg/L NAA Organogenesis Rhizome buds NA, 5 w, 2 w Agrobacterium mediated + PCR Somatic embryo formation was highest on MS medium with 5 mg/L 2,4-D and 5 mg/L NAA, reaching 87.50% in Curcuma longa and 95.83% in C. mangga. Increasing NAA or 2,4-D to 8 mg/L reduced embryo formation to 4.2% and 8.3% in C. longa, and 4.2% and 12.5% in C. mangga, respectively [106]
      36 Curcuma longa L. Management of osteoarthritis, fever, depression, liver diseases, and high cholesterol Sweden, India Callus: MS + 1 mg/L basta; shoot: 2.2 μM BA + 0.93 μM kinetin + 5% coconut water + 2% sucrose Indirect somatic embryogenesis Mature rhizomes 4 w, 3−4 w, NA Particle bombardment
      gene gun method
      + GUS + PCR      		 Transient GUS expression was observed in 91% of bombarded embryos after 24 h, while stable expression was detected in the shoot tips and roots of plantlets after 3 months [107,108]
      37 India Cotyledon: B5 media + ½MS media; shoot: B5 media + 0.5 μM BA + 2.4 μM NAA; root: B5 media + 2.4 μM NAA Mature seeds NA, 25−30 d, 2−3 w Agrobacterium mediated + GUS + semi-quantitative RT-PCR The post-transformation regeneration efficiency was 2.9%, with a confirmed transformation efficiency of 1.5% (PCR-based) and a 36.4% survival rate after hardening [109,110]
      38 Vanilla planifolia Jacks. For fever, spasms, blood clotting issues, and gastrointestinal distress India, Turkey, Indonesia Plant regeneration: 4.43 μM BA + 2.68 μM NAA Direct somatic embryogenesis Shoot Ttps NA, 45 d, 45 d Agrobacterium mediated + GUS + PCR + southern blot + northern blot The highest callus regeneration (80%) occurred in VK4, while 100% direct shoot regeneration was achieved in VK3 and VK6, with VK6 also yielding 50% rooting, 33.3% indirect shoot regeneration in VK2, and 71.4% globular-stage embryo formation in VK4. [111−113]
      39 India − − − NA, NA, NA Agrobacterium mediated + GUS + PCR + southern blot + northern blot Transformation efficiency averaged 39.4% for the nptII gene and 23.4% for GUS across three independent experiments [87]
      40 V. planifolia Jacks. For fever, spasms, blood clotting issues, and gastrointestinal distress Mexico Callus: MS + 50 mg/L cysteine HCl + 100 mg/L ascorbic acid + 0.45 μM TDZ; callus: MS + 0.45 μM TDZ, MS + 8.88 μM TDZ / 11.11 μM BA Cell suspension culture Immature seeds 4 w, NA, NA − In V. planifolia in vitro culture, B:R LEDs enhanced shoot elongation and chlorophyll synthesis during multiplication, while B LEDs promoted shoot elongation, root and leaf formation during rooting. All LED treatments increased photosynthetic pigment synthesis and achieved 100% acclimatization success [114]
      41 Malaysia Shoot: MS + 1−10 mg/L BAP, ½MS + 1 mg/L BAP; root: ½MS + 1 mg/L BAP Organogenesis Nodes, shoot tips NA, 30−45 d, 30 d − Auxin-free half-strength MS medium with 1 mg/L BAP enabled 100% rooting and was optimal for shoot propagation and early differentiation, with stem nodal segments showing high regeneration potential [115]
      42 Malaysia, Moroco Shoot + root: MS + 0.5 mg/L NAA + 1 mg/L BAP; Knudson C media (KC) + 0.5 mg/L NAA + 1 mg/L BAP; Vacin & went media (VC) + 0.5 mg/L NAA + 1 mg/L BAP Direct somatic embryogenesis Nodes NA, 2−6 w, 2−6 w − MS medium produced the highest shoot (2.37 ± 0.76 cm) and root (2.04 ± 0.95 cm) lengths with well-formed leaves and extended roots, significantly outperforming KC and VW media in V. planifolia nodal segment cultures [116,117]
      43 Brazil Shoot: double culture system media + 1, 2, 3 mg/L BAP; root: double culture system media + 1, 2, 3 mg/L IBA Organogenesis Nodes, axillary buds NA, 30, 60, 90 d, 30, 60, 90 d − DPS medium with 1 mg/L BA yielded over a 2.5-fold increase in axillary shoot multiplication compared to SS medium after 90 d, with 100% rooting in full-strength MS (without IBA) and 100% acclimatization success. [118,119]
      NA: Not available.

      Table 1. 

      Integration of gene transformations and plant tissue culture techniques in plant regenerations in spice crops with medical importance.

    • S. No. Spices Country Tissue
      cultured variety      		 Tissue culture methods Traits improvement/
      population line generations      		 Explants Ref.
      1 Elethania cardamomum Matra. India − Anther Culture Homozygous Haploid Lines Anthers [128]
      2 Crocus sativus L. India − EMS Mutation Mutant Population Lines Development Corm [129]
      3 Zingiber officinale Roscoe China Wuling Somaclonal Variations Disease Free, High Performance Growth Parameters Or Yield Rhizome [130]
      4 Z. officinale Roscoe USA Baby ginger Micropropagation Cold Hardy Ginger Lines, High Yield, Early Durations Rhizome [131]
      5 Amomum subulatum Vietnam − Shoot Tip Culture Micropropagated Population Line Generations Shoot Tips [132]
      6 Piper nigrum L. India − Somatic Embryo,
      Meristem Culture      		 Virus Free Population Lines Enhancement Shoot Tips [133]
      7 E. cardamomum Matra. India − Micropropagation Virus or Disease Free Resistance Lines Such As Katte, Kokke Kundu, Nilgiri Necrosis, High Yield Population Lines Floral Buds [134]
      8 Z. officinale Roscoe India − Somaclonal Variations, Invitro Pollinations,
      Embryo Rescue      		 High Yield, High Quality Lines, Rhizome Rot or Bacterial Wilt Resistance Seed-Embryo [134]
      9 Vanilla planifolia Jacks. India − Shoot Tip Culture,
      Root Tip Culture      		 Root Rot Resistance Population Lines Development Shoot Tips, Root Tips [134]
      10 Z. officinale Roscoe, V. planifolia Jacks., P. nigrum, E. cardamoum, Cuminum cyminum India − Synthetic Seeds Artificial Seed Productions Somatic Embryo, Shoot Tips [134]
      11 Z. officinale Roscoe Malaysia − Micropropagation-

      Rhizome Culture

      		 Disease Free Plants, Secondary Metabolites or Anti-Oxidants Synthesis Immature Rhizome Buds [135]
      12 Z. officinale Roscoe China − Root Tip Culture High Yield, Autotetraploid Population Lines Generations, High Gingerol Contents Root Tip [136]
      13 Curcuma longa L. India − Micropropagation High Yield, Rhizome Rot Resistance, Uniform Population Lines, High Curcumin Contents Rhizome [137]
      14 Allium sativum L. Philippines − Shoot Tip Culture Onion Yellow Dwarf Virus, Leek Yellow Strip Potyvirus Resistant Lines, High Yield Medium Size Bulb, Shoot Tips [138]

      Table 2. 

      Generations of population lines and variety in spice crops with plant tissue culture techniques.

    • S. No. Spices Country Media + growth
      regulator levels      		 Tissue culture methods Explants Callus induction, shoot regeneration, root regeneration (weeks) Transformation methods Regeneration and transformation efficiency Ref.
      1 Trigonella foenum-graecum L. Greece Callus + root: 2.2 g/L MS salts + vitamins + 1% sucrose + 0.05% MES (2-N-morpolino ethanesulfonic acid); full strength McCowns WPM + 3% sucrose + 0.05% MES; full strengths Hoagland nutrient solutions Hairy root culture/Indirect organogenesis Seeds 2, NA, 2.5−3 Agrobacterium rhizogenes
      + GFP + PCR      		 Callus responder transformed root at 12 d in solid or liquid media [159]
      2 Coriandrum sativum L. India Callus: MS + 1 mg/L 2,4 D; embryo: MSH media (Murashige & Skoog, 1962 and Schenk and Hildebrandt, 1972) + 0.5 mg/L NAA + 1 mg/L 2,4 D; shoot: MSH media + 0.5 mg/L BAP Indirect somatic embryogenesis Seed: radicle: root tip NA, NA, NA Agrobacterium tumefaciens + GUS + PCR Embryo successfully exhibited plantlets [160]
      NA: Not available.

      Table 3. 

      Approaches of genetic transformations and plant tissue culture techniques in non-chimeric plant regenerations in home garden spice crops.