Abstract
Kinnow ( Citrus nobilis Lour. × Citrus deliciosa Ten.) needs to be genetically improved for traits such as seedlessness using biotechnological tools. Indirect somatic embryogenesis (ISE) protocols have been reported for citrus improvement. However, its use is restricted due to frequent occurrences of somaclonal variation and low recovery of plantlets. Direct somatic embryogenesis (DSE) using nucellus culture has played a significant role in apomictic fruit crops. However, its application in citrus is limited due to the injury caused to tissues during isolation. Optimization of the explant developmental stage, explant preparation method, and modification in the in vitro culture techniques can play a vital role in overcoming the limitation. The present investigation deals with a modified in ovulo nucellus culture technique after the concurrent exclusion of preexisting embryos. The ovule developmental events were examined in immature fruits at different stages of fruit growth (stages I–VII). The ovules of stage III fruits (>21–25 mm in diameter) were found appropriate for in ovulo nucellus culture. Optimized ovule size induced somatic embryos at the micropylar cut end on induction medium containing Driver and Kuniyuki Walnut (DKW) basal medium with kinetin (KIN) 5.0 mg L -1 and malt extract (ME) 1,000 mg L -1 . Simultaneously, the same medium supported the maturation of somatic embryos. The matured embryos from the above medium gave robust germination with bipolar conversion on Murashige and Tucker (MT) medium + gibberellic acid (GA 3 ) 2.0 mg L -1 + ά-naphthaleneacetic acid (NAA) 0.5 mg L -1 + spermidine 100 mg L -1 + coconut water (CW) 10% (v/v). The bipolar germinated seedlings established well upon preconditioning in a plant bio regulator (PBR)-free liquid medium under the light. Consequently, a cent percent survival of emblings was achieved on a potting medium containing cocopeat:vermiculite:perlite (2:1:1). Histological studies confirmed the single nucellus cell origin of somatic embryos by undergoing normal developmental events. Eight polymorphic Inter Simple Sequence Repeats (ISSR) markers confirmed the genetic stability of acclimatized emblings. Since the protocol can induce rapid single-cell origin of genetically stable in vitro regenerants in high frequency, it has potential for the induction of solid mutants, besides crop improvement, mass multiplication, gene editing, and virus elimination in Kinnow mandarin.
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Claims 8
Claim · model-written summary
Stage III fruit ovules were appropriate for in ovulo nucellus culture.
Verbatim
e0“The ovules of stage III fruits (>21–25 mm in diameter) were found appropriate for in ovulo nucellus culture.”
t0“Kinnow mandarin”
Claim · model-written summary
Optimized ovules induced somatic embryos on DKW medium with KIN and malt extract.
- Taxon
- Kinnow mandarint0
- Organ / stage
- ovulee0
- Treatment
- Driver and Kuniyuki Walnut (DKW) basal medium with kinetin (KIN) 5.0 mg L -1 and malt extract (ME) 1,000 mg L -1e0
- Outcome
- somatic embryos at the micropylar cut ende0
Not reportedComparatorDirection
Verbatim
e0“Optimized ovule size induced somatic embryos at the micropylar cut end on induction medium containing Driver and Kuniyuki Walnut (DKW) basal medium with kinetin (KIN) 5.0 mg L -1 and malt extract (ME) 1,000 mg L -1 .”
t0“Kinnow mandarin”
Claim · model-written summary
The induction medium also supported somatic embryo maturation.
Verbatim
e0“Simultaneously, the same medium supported the maturation of somatic embryos.”
t0“Kinnow mandarin”
Claim · model-written summary
Mature embryos germinated with bipolar conversion on MT medium with growth regulators and coconut water.
- Taxon
- Kinnow mandarint0
- Organ / stage
- matured embryose0
- Treatment
- Murashige and Tucker (MT) medium + gibberellic acid (GA 3 ) 2.0 mg L -1 + ά-naphthaleneacetic acid (NAA) 0.5 mg L -1 + spermidine 100 mg L -1 + coconut water (CW) 10% (v/v)e0
- Outcome
- robust germination with bipolar conversione0
Not reportedComparatorDirection
Verbatim
e0“The matured embryos from the above medium gave robust germination with bipolar conversion on Murashige and Tucker (MT) medium + gibberellic acid (GA 3 ) 2.0 mg L -1 + ά-naphthaleneacetic acid (NAA) 0.5 mg L -1 + spermidine 100 mg L -1 + coconut water (CW) 10% (v/v).”
t0“Kinnow mandarin”
Claim · model-written summary
Bipolar germinated seedlings established well after preconditioning in regulator-free liquid medium under light.
- Taxon
- Kinnow mandarint0
- Treatment
- preconditioning in a plant bio regulator (PBR)-free liquid medium under the lighte0
- Outcome
- established welle0
Not reportedComparatorDirection
Verbatim
e0“The bipolar germinated seedlings established well upon preconditioning in a plant bio regulator (PBR)-free liquid medium under the light.”
t0“Kinnow mandarin”
Claim · model-written summary
Emblings had 100 percent survival on cocopeat, vermiculite, and perlite potting medium.
Verbatim
e0“Consequently, a cent percent survival of emblings was achieved on a potting medium containing cocopeat:vermiculite:perlite (2:1:1).”
t0“Kinnow mandarin”
Claim · model-written summary
Histology confirmed somatic embryos originated from a single nucellus cell and underwent normal development.
Verbatim
e0“Histological studies confirmed the single nucellus cell origin of somatic embryos by undergoing normal developmental events.”
t0“Kinnow mandarin”
Claim · model-written summary
ISSR markers confirmed the genetic stability of acclimatized emblings.
Verbatim
e0“Eight polymorphic Inter Simple Sequence Repeats (ISSR) markers confirmed the genetic stability of acclimatized emblings.”
t0“Kinnow mandarin”
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